JP5815638B2 - Composition for MGD-CSF in disease treatment and method of use thereof - Google Patents

Abstract

The present invention relates to in vitro methods for stimulating, or increasing a population of, immune cells comprising using compositions which comprise a substantially pure polypeptide comprising a first amino acid sequence having at least about 70% homology to a sequence selected from SEQ. ID. NOs:7, 8, 9, and 11; or a biologically active fragment of any of these, wherein said polypeptide or fragment thereof can stimulate the proliferation and differentiation of granulocytes, monocytes, or dendritic cells; and to the use of these polypeptides in methods of treatment

Description

(Claiming priority)
This application is related to provisional application 60 / 590,565 filed July 22, 2004; 60 / 647,604 filed January 27, 2005; 60 application filed March 24, 2005. And "Novel MGD-CSF Polypeptides, Polynucleotides, and Methods" filed Jul. 14, 2005;
claims priority to the provisional application entitled “of USe Thereof”. This application also includes PCT / US03 / 34811 filed Oct. 31, 2003; PCT / US04 / 11270 filed Apr. 30, 2004; No. 60 / 590,565 filed Jul. 22, 2004. No. 60 / 642,604, filed Jan. 11, 2005; No. 60 / 647,013, filed Jan. 27, 2005; and No. 60 / 654,229, filed Jan. 18, 2005; Regarding application. These applications are fully incorporated by reference in their entirety.

(Technical field)
The present invention relates to a novel secreted molecule identified herein as “monocytes, granulocytes, and dendritic cell colony stimulating factor” (MGD-CSF). . The present invention relates to MGD-CSF polypeptides and polynucleotide sequences, fusion molecules comprising MGD-CSF, vectors, host cells, compositions and kits comprising MGD-CSF, as well as immune related diseases, infectious diseases and cancer. It relates to methods of using MGD-CSF and related molecules to diagnose, prevent, confirm and treat prognosis of diseases including. MGD-CSF is a splicing variant of the gene MCG34647 whose function is previously unknown.

(Background of the Invention)
Cells of the innate immune system, such as monocytes, macrophages, natural killer (NK) cells, and polymorphonuclear neutrophils (PMN), are phagocytic, cytolytic and antibacterial. Is a first-line defense against cancer and infectious diseases. Monocytes and macrophages are thought to play an important role in inflammatory diseases through the activation and secretion of inflammatory mediators. For example, granulocyte macrophage-colony stimulating factor (GM-CSF) is known to promote the proliferation and differentiation of granulocytes, monocytes and macrophages. Granulocyte-colony stimulating factor (G-CSF) is known to promote differentiation and proliferation of granulocytes and neutrophils (Non-patent Document 1). Currently, both G-CSF and GM-CSF are used as protein therapeutics to promote blood cell recovery after chemotherapy, radiation therapy and bone marrow transplantation.

  NK cells are known to play a role in the host response to cancer. In both syngeneic and xenograft graft models, tumor cells proliferate more efficiently in NK − / − mice, and the survival rate of mice in these models is similar to that for mice bearing NK cells. Significantly lower than survival rate. Furthermore, enhancement of the NK response with soluble protein factors such as IL-2 or IL-15 increases the efficiency of NK cells to kill tumor cells in the presence of anti-tumor antibodies, both in vitro and in vivo. Is shown (Non-Patent Document 2).

  Furthermore, NK cells are also known to play a role in host responses to infectious diseases. For example, mice lacking NK cells are known to have increased sensitivity to viruses and intracellular pathogens. Similarly, humans with naturally occurring deletions of NK cells are known to be extremely sensitive to infection. In vitro, NK-mediated killing of cells infected with viruses or other intracellular pathogens is known to be enhanced by cytokines such as interferon alpha, interferon beta, interleukin 12 and interleukin 18 (non- Patent Document 3); Non-Patent Document 4; Non-Patent Document 5).

  It is also known that activated NK cells can correlate with the failure rate of women undergoing in vitro fertilization (IVF) procedures and can be further associated with pregnanity loss (Non-patent document 6). Furthermore, elevated levels of activated NK cells can be found in a number of patients with immune endometriosis, known as a background cause of female infertility (6).

  Dendritic cells that process antigens can sensitize T cells to both novel and recall antigens. Dendritic cells express high levels of major histocompatibility complex class I and II antigens, which, along with other immunoregulatory proteins, adhesion factors and cytokines, play a role in cancer immunotherapy. Dendritic cell cancer vaccines have been reported to be generated by extracting patient dendritic cells and regenerating large numbers of dendritic cells in vitro or ex vivo using immune cell stimulators. The dendritic cells can then be exposed to an antigen derived from the patient's cancer cells. A combination of dendritic cells and antigen is injected into the patient, and the dendritic cells program the patient's T cells. Dendritic cells destroy antigens on the surface of cancer cells and then present them to killer T cells (7).

  Cancer patients recovering from autologous hematopoietic cell transplantation show reduced levels of circulating dendritic cells. Dendritic cells develop from hematopoietic progenitors, and the promotion of their development can help reacquire normal dendritic cell levels. The ability to generate dendritic cells by inducing the proliferation of isolated human dendritic cells and inducing the proliferation and differentiation of hematopoietic stem cells facilitates the efficacy testing of dendritic cell vaccination, And effective vaccination implementation becomes easy. There is a need in the art for factors that stimulate dendritic cell proliferation and / or hematopoietic stem cell proliferation and / or differentiation into dendritic cells. There is also a need in the art for factors that promote the generation of dendritic cells from hematopoietic cells in order to increase circulating dendritic cells in a preparation for hematopoietic cell transplantation.

  Osteoclasts share a common precursor with dendritic cells, macrophages and microglia (Non-patent Document 8). These multi-faceted, adherent, bone-resorbing cells function in the vicinity of bone to differentiate in the bone marrow and regulate bone remodeling and calcium homeostasis. It has been reported that osteoclast differentiation and function are regulated by secretory factors including M-CSF and osteoprotegerin (RANK ligand) (Non-patent Document 9). Factors that play a role in the regulation of osteoclast differentiation and function may be therapeutic in treating osteoporosis and other bone diseases.

  Microglial cells function as immune effectors of the central nervous system, and they also produce neurotrophic factors to regulate glutamate uptake. These mononuclear phagocytes are distributed throughout the parenchyma of the central nervous system in both white and gray matter. Microglia are present in increased numbers in patients with Alzheimer's disease, where microglia are reported to show nitric oxide production and a marked increase in inflammatory cytokines including IL-1 and MIP1α (non-patented). Reference 10). Factors that regulate microglia differentiation and function include Alzheimer's disease and other demyelinating diseases such as multiple sclerosis, acute disseminated encephalomyelitis, progressive multifocal leukoencephalopathy, stroke and Parkinson's disease It may be therapeutic to treat neurological diseases.

  The gene MGC34647 encodes a hypothetical protein NP-689669 (Non-patent Document 11). The function of this gene and its encoded polypeptide has previously been unknown. The sequences of MGC34647 and NP_6869669 correspond to SEQ ID NO: 49 and SEQ ID NO: 103 of Patent Document 1, respectively. They respectively correspond to the amino acid sequence of the secreted protein whose function is unknown and its coding sequence.

International Publication No. 2002/048337 Pamphlet

Ogawa, Blood, 1993, 81, p. 3844 to 2853 Carson et al. Exp. Med. 1994, 180, p. 1395 to 1403 Wu et al., Adv. Cancer Res. 2003, volume 90, p. 127 Biron et al., Rev. Immunol. 1999, Vol. 17, p. 189 Naume et al., Scand. J. et al. Immunol. 1994, 40, p. 128 Dosou et al., Endocr. Rev. 2005, Vol. 26, p. 44 Song et al., Yonsei Med. J. et al. 45, 2004, Addendum, p. 48-52 Servet-Delpra et al., BMC Immunol. 2003, Vol. 3, p. 15 Miyamoto et al., Keio. J. et al. Med. 2003, vol. 52, p. 1-7 Vincent et al., Neurobiol. Aging, 2002, Vol. 23, p. 349-362 Straussberg et al., Proc. Natl. Acad. Sci. 2002, vol. 99, p. 16, p899

(Industrial utility)
Current treatments for immune diseases, cancer, infectious diseases and immune-mediated recurrent miscarriages are inadequate, inadequate and often toxic. New therapeutic compounds and agents that increase the effectiveness of the innate immune response against these conditions can provide more thorough treatment and may have a better therapeutic index than current therapeutic agents.

(Short description of the table)
Table 1 shows information on SEQ ID NOs: 1-271 listed in the sequence listing. The first column indicates an internal designation identification number (FP ID). The second column shows the nucleotide sequence identification number (SEQ ID NO: (N1)) of the open reading frame of the nucleic acid sequence (SEQ ID NO: 1). The third column shows the amino acid sequence identification number (SEQ ID NO: (P1)) of the polypeptide sequence. The fourth column shows the nucleotide sequence identification number (SEQ ID NO: N0) of the entire nucleic acid sequence, including coding and non-coding regions. The fifth column shows the corresponding naming or NCBI accession number (Source ID). The sixth column indicates the type of sequence, eg, function or vector composition (Type).

Table 2 annotates MGD-CSF to NP-689669, a publicly disclosed sequence with the greatest degree of similarity. The first line shows an internal designation identification number (FP ID). The second row shows the clone identification number (Clone ID). The third line shows the predicted length of the polypeptide in number of amino acid residues (Pred Prot Len). The fourth row shows the top human hit accession number (Top Human Hit Accession No) found in the NCBI public database. Line 5 shows the top human hit annotation shown in line 4 (Top Human Hit Annotation). Line 6 shows the length of this top human hit in the number of amino acid residues (Top Human Hit Len). Line 7 shows the length of the match (Match Len) in the number of amino acid residues between the query sequence indicated by the FP ID and the top human hit. Line 8 shows the percent identity between the FP ID spanning the length of the expressed FP ID amino acid sequence and the top human hit as a percentage (Top Human Hit ID% across Query Len (Top Human Hit ID%). % ID over
Query Len)). Line 9 shows the percent identity between the FP ID spanning the length of the top human hit and the top human hit (ID% across Human Hit Len) (% ID over Human Hit Len).

  Table 3 shows the protein arrangement of MGD-CSF and NCBI NP_689669. The first row shows the designated internal ID number (FP ID) of the polypeptide. The second line shows the clone identification number or the NCBI accession number (Clone ID) of the polypeptide. The third line shows the internal cluster identification number (CLaster) of this polypeptide. The fourth line shows that NP-689669 is secreted (Classification). Line 5 shows the predicted protein length in number of amino acid residues (Pred Prot Len). Line 6 shows an internal parameter that predicts the probability of FP ID being secreted, where “1” is likely to secrete this polypeptide and “0” is secretable. Low nature (Treevote). The seventh line shows the position (Signal Peptide Codes) of the signal peptide. The eighth line shows the protein arrangement of the mature polypeptide having the first amino acid residue at the N-terminus of the full-length polypeptide having 1 amino acid (Mature Protein Coords).

  Table 4 shows the annotation of the secretory leader sequence shown in Table 1. The first column shows the internal ID number (FP ID) inside this polypeptide. The second column shows a reference identification number (Source ID). The third column shows the NCBI annotation for this sequence.

  Table 5 provides annotations for the MGD-CSF construct shown in Table 1. The first column shows the clone identification number (Clone ID). The second column shows NCBI annotation (Annotation). The third column shows a list of vectors (Vector Description). The fourth column lists the tags, if any (Tag).

  Table 6 shows the effect of the MGD-CSF construct on bone marrow cell proliferation in vitro as further described in Example 9C. The first column shows the clone identification number (Clone ID). The second column shows the amino acid sequence of this clone. The third row shows a semiquantitative depiction (potency) of the titer of activity of each clone that stimulates monocyte proliferation. The fourth line shows a semi-quantitative expression of the degree of expression of each construct.

  Table 7 shows the effect of MGD-CSF construct on bone marrow cell proliferation in vivo. Table 7A shows the results of injection into a group of 6 mice, with 3 as control vectors and 3 as human MGD-CSF constructs for bone marrow cell proliferation. Table 7B shows the results of injection into a group of 12 mice, 6 as control vectors and 6 as mouse MGD-CSF. In both Table 7A and Table 7B, the first column lists the animal identification number (Animal ID), the second column lists the vector as control or MGD-CSF (Description), and the third column Indicates the number of monocytes in the peripheral blood (monocytes / μl).

Table 8 shows GeneLogic using an Affymetrix U133 tip probe.
The expression of MGD-CSF gene determined by querying the database is shown. The first column lists diseases in which MGC34647 was overexpressed (Disease). The second column lists the specific pathologies associated with the diseases in the first column (Pathology). The third column shows the number of disease types tested positive for the presence of MGC34647 (MGC34647 positive). Column 4 lists the number of specimens examined (Total Gene Logic). The fifth column lists the percentage of tested specimens that were positive (% Total). Columns 6 and 7 show that three acute promyelocytic leukemia samples (13% of the total number tested) were derived from bone marrow.

(Summary)
MGD-CSF promotes proliferation, survival and / or differentiation of monocytes, granulocytes, dendritic cells and NK cells. Thus, MGD-CSF is a protein therapy for treating cancer through its ability to stimulate the proliferation and / or activation of immune cells such as NK cells, monocytes, macrophages, granulocytes and dendritic cells that fight tumor cells. Find uses as agents. MGD-CSF can be used alone or in combination with therapeutic monoclonal antibodies (eg, Rituxan) to treat cancer. This is because MGD-CSF can promote antibody-dependent cytotoxicity mediated by NK cells, monocytes or granulocytes. MGD-CSF can also be used as an antagonistic therapeutic protein to perform adjuvant hematopoietic regeneration for chemotherapy and bone marrow transplantation. This can further be used to increase the number of dendritic cells in vivo or ex vivo. Furthermore, MGD-CSF may be useful as an anti-infectious agent in the treatment of infectious diseases such as those caused by bacteria or viruses (eg, hepatitis C virus (HCV) or human immunodeficiency virus (HIV)). .

  MGD-CSF promotes immune cell proliferation and / or differentiation and thus finds use in the treatment of immune diseases. This may play a role in the development and treatment of autoimmune diseases. MGD-CSF antagonists can be developed as therapeutic agents for treating immune diseases. Antagonists can include monoclonal antibodies to MGD-CSF; MGD-CSF receptors including soluble receptors; nonfunctional mutants; antisense DNA; and RNAi.

  The present invention comprises a first polynucleotide comprising a first nucleotide sequence selected from SEQ ID NO: 1, 2, 3 and 5; comprising a first amino acid sequence selected from SEQ ID NO: 7, 8, 9 and 11 A first polynucleotide encoding the first polypeptide; a polynucleotide comprising a nucleotide sequence complementary to the first nucleotide sequence; and an isolated comprising any biologically active fragment thereof. Nucleic acid molecules are provided. In certain embodiments, the biologically active polypeptide fragment comprises at least 6 consecutive amino acid residues selected from SEQ ID NOs: 7, 8, 9, and 11, wherein the 6 consecutive amino acid residues At least two of these are leucine and arginine residues at amino acid residue positions 80 and 81, respectively. The isolated nucleic acid molecule can be selected from cDNA molecules, genomic DNA molecules, cRNA molecules, siRNA molecules, RNAi molecules, mRNA molecules, antisense molecules and ribozymes. In certain embodiments, the nucleic acid molecule further comprises its complement.

  In certain embodiments, the first nucleotide sequence is SEQ ID NO: 3. This embodiment may further comprise a second polynucleotide. This second polynucleotide may comprise a second nucleotide sequence encoding a homologous or heterologous secretory leader. This secretory leader can be selected from SEQ ID NOs: 14-211.

  The invention also provides nucleic acid molecules that are at least about 70%, at least about 80%, or at least about 90% identical to the isolated nucleic acid molecules described above. The present invention specifically hybridizes under stringent conditions to the sequences shown in SEQ ID NOs: 1, 2 or 3 or to the complements of the sequences shown in SEQ ID NOs: 1, 2, 3 and 5. An isolated nucleic acid molecule, wherein the nucleic acid molecule encodes a polypeptide capable of stimulating the proliferation and differentiation of granulocytes, monocytes and dendritic cells is provided.

  The invention further comprises a first amino acid sequence selected from SEQ ID NO: 7, 8, 9 and 11; a sequence encoded by SEQ ID NO: 1, 2, 3 and 5; An isolated polypeptide comprising an active fragment is provided. The isolated polypeptide can be present in a cell culture, such as a bacterial cell culture, a mammalian cell culture, an insect cell culture, or a yeast cell culture, or in a cell culture medium. The isolated polypeptide can also be present in a plant or non-human animal.

  In certain embodiments, the biologically active fragment comprises at least 6 consecutive amino acid residues selected from SEQ ID NOs: 7, 8, 9, and 11 and at least of the 6 consecutive amino acid residues. Two are leucine and arginine at amino acid residues 80 and 81 of SEQ ID NO: 5.

  The invention further provides at least about 70%, at least about 80%, or at least about 90% homology to an isolated polypeptide comprising a first amino acid sequence selected from SEQ ID NOs: 7, 8, 9, and 11. An isolated polypeptide; the sequences encoded by SEQ ID NOs: 1, 2, 3, and 5; and biologically active fragments of any of these.

  The present invention relates to an isolated polypeptide comprising a first amino acid sequence selected from SEQ ID NO: 7, 8, 9 and 11; a sequence encoded by SEQ ID NO: 1, 2, 3 and 5; and any of these A biologically active fragment further comprising a second amino acid sequence, wherein the second amino acid sequence is a homologous or heterologous secretory leader, the first and second A fragment is provided wherein the amino acid sequences of are operably linked. This secretory leader sequence can be selected from SEQ ID NOs: 14-211.

  The present invention also provides a vector comprising the above nucleic acid molecule and a promoter that regulates its expression. This vector may be a viral vector or a plasmid vector. The promoter may be naturally adjacent to the nucleic acid molecule or may not be naturally adjacent to the nucleic acid molecule. This may be an inducible promoter, a condition activated promoter, a constitutive promoter and / or a tissue specific promoter.

  Furthermore, the present invention provides a recombinant host cell comprising the cell and one or more isolated nucleic acids, polypeptides or vectors as described above. The host cell may be a prokaryotic cell or a eukaryotic cell, such as a human cell, a non-human mammalian cell, an insect cell, a fish cell, a plant cell or a fungal cell. In certain embodiments, the mammalian cell is a CHO cell line or a 293 cell line, such as a 293T cell or a 293E cell.

  The invention further provides non-human animals injected with the isolated nucleic acid or polypeptide of the invention. The animal may be, for example, a rodent, a non-human primate, a rabbit, a dog or a pig.

  The present invention still further provides a nucleic acid composition comprising an isolated nucleic acid molecule of the present invention and a carrier. The present invention provides a polypeptide composition comprising an isolated polypeptide of the present invention and a carrier. The present invention provides a vector composition comprising the vector of the present invention and a carrier. The present invention provides a host cell composition comprising a host cell of the present invention and a carrier. In certain embodiments, the carrier is a pharmaceutically acceptable carrier.

  In another aspect, the present invention provides a method for producing a recombinant host cell comprising providing a vector comprising an isolated nucleic acid molecule of the present invention, and contacting the cell with the vector. Forming a recombinant host cell transfected with the nucleic acid molecule.

  The present invention provides a method of producing a polypeptide comprising the steps of providing an isolated nucleic acid molecule of the present invention and expressing the nucleic acid molecule in an expression system to produce a polypeptide. Is included. In certain embodiments, the expression system is a cell expression system, such as a prokaryotic expression system or a eukaryotic expression system. The expression system may include a host cell transfected with an isolated nucleic acid molecule of the present invention that forms a recombinant host cell, and further comprising culturing the recombinant host cell to produce a polypeptide. Is included. In certain embodiments, the expression system is a wheat germ lysate, rabbit reticulocyte, ribosome display and E. coli. A cell-free expression system derived from E. coli lysate.

  The invention also provides a polypeptide produced by such a method. For example, the present invention provides a polypeptide produced by an eukaryotic expression system as described above, wherein the host cell is selected from a mammalian cell, insect cell, plant cell, yeast cell and bacterial cell. provide.

  In yet another aspect, the present invention provides a diagnostic kit comprising a composition comprising an isolated nucleic acid molecule of the present invention, a reporter for detecting the nucleic acid molecule or its complement, and a vehicle. The present invention provides a diagnostic kit comprising an antibody that specifically binds to an isolated polypeptide of the present invention and a carrier. The invention also provides a diagnostic kit comprising an isolated polypeptide of the invention and a carrier.

  In a further aspect, the invention provides a method for detecting the presence of an antibody specific for an isolated polypeptide of the invention in a patient sample, the method comprising an isolated polypeptide of the invention. Providing a composition; allowing the polypeptide to interact with the sample; and determining, if present, whether an interaction has occurred between the polypeptide and an antibody in the sample. Including.

In yet a further aspect, the present invention specifically binds to and / or interferes with the activity of the antigen comprising at least 6 consecutive amino acid residues selected from SEQ ID NOs: 7-12. An isolated antibody is provided. For example, these consecutive amino acid residues may include the conserved amino acid residue leu-arg at amino acid positions 80 and 81 of SEQ ID NO: 7, or the conserved amino acid residue leu-gln-arg of SEQ ID NO: 12. This antibody, polyclonal antibody, monoclonal antibody, single chain antibody and any active fragments thereof, for example, antigen-binding fragment, Fc fragment, cdr fragment, V _H fragments can be selected from V _C fragment and framework fragments .

  The present invention also provides an isolated polypeptide as described above further comprising at least one fusion partner. As an example, the fusion partner can be selected from a polymer, polypeptide, succinyl group, fetuin A, fetuin B, leucine zipper domain, tetranectin trimerization domain, mannose binding protein and Fc region. The polymer can be, for example, a polyethylene glycol moiety that can be attached via the amino group of an amino acid of a polypeptide. The polyethylene glycol moiety may be a branched polymer or a linear polymer.

  The present invention further provides a method of screening for factors that modulate the activity of an isolated polypeptide of the present invention, which test that the isolated polypeptide of the present invention affects biological activity. Providing a system; screening a plurality of factors for an effect on the activity of the isolated polypeptide of the invention in the test system. This modifier may be, for example, a small molecule drug. This modifier may also be an antibody, for example.

  The present invention further provides a method of stimulating immune cells, which provides a composition comprising a substantially pure polypeptide selected from any of claims 7-12 and an active fragment thereof. And contacting the polypeptide with one or more immune cells. This polypeptide can be encoded by a nucleic acid molecule comprising a nucleotide sequence selected from SEQ ID NOs: 1-6. Suitable immune cells include granulocytes; monocytes; lymphocytes such as NK cells; macrophages; peripheral blood mononuclear cells; and dendritic cells.

  The present invention still further provides a method of increasing the population of immune cells, the method comprising a substantially pure polypeptide selected from SEQ ID NOs: 7-12 and any active fragment thereof. Providing an article; contacting the polypeptide with one or more immune cells or immune cell precursors. This polypeptide can be encoded by a nucleic acid molecule comprising a nucleotide sequence selected from SEQ ID NOs: 1-6. Suitable immune cell populations include granulocytes; monocytes; lymphocytes such as NK cells; macrophages; and peripheral blood mononuclear cell populations.

  The invention further provides a method of stimulating an immune response in a subject, the method comprising a substantially pure polynucleotide encoding a polypeptide selected from SEQ ID NOs: 7-12 and any activity thereof Providing a composition comprising such a fragment; and administering the composition to the subject. This polypeptide can be encoded by a nucleic acid molecule comprising a nucleotide sequence selected from SEQ ID NOs: 1-6. The polypeptide may be administered locally or systemically. It can be administered intravenously, by enema, intraperitoneally, subcutaneously, topically or transdermally.

  The present invention provides a method of expanding immune cells in a subject undergoing cancer therapy, comprising a substantially pure polypeptide selected from any of SEQ ID NOs: 7-12 and those Providing a composition comprising any of the active fragments; and administering the composition to the subject. This polypeptide can be encoded by a nucleic acid molecule comprising a nucleotide sequence selected from SEQ ID NOs: 1-6. Suitable immune cell populations include monocytes; lymphocytes such as NK cells; macrophages; and peripheral blood mononuclear cell populations. Cancer treatment may or may include chemotherapy and / or radiation therapy. The polypeptide may be administered after bone marrow transplantation.

  The present invention also provides a method of treating or preventing cancer in a subject, comprising a substantially pure polypeptide selected from SEQ ID NOs: 7-12 and any active fragment thereof. Providing a composition; and administering the composition to the subject.

  The present invention further provides a method of inhibiting tumor growth in a subject, the method comprising a substantially pure polypeptide selected from SEQ ID NOs: 7-12 and any active fragment thereof. Providing a product and administering the composition to the subject. The tumor can include human tumor cells, such as solid tumor cells or leukemia tumor cells.

  The present invention still further provides a method of treating or preventing infection in a subject, comprising: a substantially pure polypeptide selected from SEQ ID NOs: 7-12 and any active fragment thereof. Providing a composition comprising; and administering the composition to the subject. This polypeptide can be encoded by a nucleic acid molecule comprising a nucleotide sequence selected from SEQ ID NOs: 1-6. This method can treat or prevent bacterial infections, mycoplasma infections, fungal infections, viral infections, intracellular pathogens and / or intracellular parasites, for example. This method can be performed by administering the composition locally or systemically to the subject.

Furthermore, the invention provides a method of modulating an immune response in a subject, the method providing a modulator of a polypeptide selected from SEQ ID NOs: 7-12 and any active fragment thereof. And administering the modifier to the subject. This modifier may be, for example, an antibody, a soluble receptor, and / or a polypeptide. Suitable antibody modifiers, monoclonal antibodies, polyclonal antibodies, cdr fragment, V _H fragments, V _C fragment, framework fragments, active fragments of single chain antibodies and antibodies. This modifier may also be, for example, an aptamer, RNAi, an antisense molecule, and / or a ribozyme. This method may modulate the immune response, for example, by suppressing inflammatory and / or autoimmune diseases. This method may also modulate the immune response, for example by treating or preventing rheumatoid arthritis, osteoarthritis, psoriasis, inflammatory bowel disease, multiple sclerosis, myocardial infarction, stroke and / or fulminant liver failure .

  The present invention provides a method of modulating an immune response to pregnancy, the method comprising providing a polypeptide selected from SEQ ID NOs: 7-12, and modulators of any active fragments thereof; Administering the modifier to the subject. This method can, for example, modulate the immune response to reduce recurrent miscarriage.

  The present invention provides a method of enhancing an immune response to a vaccine in a subject, comprising a substantially pure polypeptide selected from SEQ ID NOs: 7-12 and any active fragment thereof. Providing a polypeptide composition; providing a vaccine composition; and administering the polypeptide composition and the vaccine composition to the subject. The polypeptide composition can be administered to the subject prior to, substantially simultaneously with, or after administering the vaccine composition.

The invention also provides a method of treating or preventing an inflammatory disease in a subject, which method provides a modulator of a polypeptide selected from SEQ ID NOs: 7-12 and any active fragment thereof. And administering the modifier to the subject. This modifier may be, for example, an aptamer, RNAi, an antisense molecule, and / or a ribozyme. This modifier may also be, for example, an antibody, a soluble receptor, and / or a polypeptide. Suitable antibody modifiers, monoclonal antibodies, polyclonal antibodies, cdr fragment, V _H fragments, V _C fragment, framework fragments, active fragments of single chain antibodies and antibodies.

The present invention further provides a method of treating or preventing an autoimmune disease in a subject comprising: a polypeptide selected from SEQ ID NOs: 7-12, and a modifier of any active fragment thereof. Providing; and administering the modifier to the subject. This modifier may be, for example, an aptamer, RNAi molecule, antisense molecule, and / or ribozyme. This modifier may also be, for example, an antibody, a soluble receptor and / or a polypeptide. Suitable antibody modifiers, monoclonal antibodies, polyclonal antibodies, cdr fragment, V _H fragments, V _C fragment, framework fragments, active fragments of single chain antibodies and antibodies.

  The present invention still further provides a method of increasing the number of NK cells in a subject, the method providing a polypeptide selected from SEQ ID NOs: 7-12, and any active fragment thereof. And administering the polypeptide to the subject.

The present invention provides a method of modulating an NK cell population in a subject, the method providing a polypeptide selected from SEQ ID NOs: 7-12, and modulators of any active fragments thereof. And administering the modifier to the subject. This modifier may be, for example, an aptamer, RNAi molecule, antisense molecule, and / or ribozyme. This modifier may also be, for example, an antibody, a soluble receptor and / or a polypeptide. Suitable antibody modifiers, monoclonal antibodies, polyclonal antibodies, cdr fragment, V _H fragments, V _C fragment, framework fragments, active fragments of single chain antibodies and antibodies. In certain embodiments, the NK cell population stimulates an immune response. In certain embodiments, the NK cell population suppresses miscarriage. In certain embodiments, the NK cell population stimulates an anticancer response.

  The invention also provides a method of expanding a population of hematopoietic stem cells, the method comprising a substantially pure polypeptide selected from SEQ ID NOs: 7-12, and an active fragment of any of them. Providing a product; contacting the population of hematopoietic stem cells with the polypeptide.

  The present invention further provides a method of providing a cytoprotective effect on a population of cells, the method comprising a substantially pure polypeptide selected from SEQ ID NOs: 7-12, and any activity thereof Providing a composition comprising such fragments; and contacting the population of cells with the polypeptide.

Methods for modulating or enhancing an immune response, treating or preventing a disease, increasing the number of NK cells, modulating a population of NK cells, expanding a population of hematopoietic stem cells, and providing cytoprotection include: For example, it can be done by administering the above composition locally or systemically. They also provide a polypeptide composition as described above, wherein the polypeptide is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from SEQ ID NOs: 1-6, and any active fragments thereof. Can be done by doing. They may further be performed by providing a polypeptide composition as described above, wherein the polypeptide further comprises at least one fusion partner. For example, the fusion partner may be selected from a polymer, polypeptide, succinyl group, fetuin A, fetuin B, leucine zipper domain, tetranectin trimerization domain, mannose binding protein and Fc region. The polymer may be a polyethylene glycol moiety, which may be attached, for example, via the amino group of an amino acid of the polypeptide. The polyethylene glycol moiety may be a branched polymer or a linear polymer.
For example, the present invention provides the following items.
(Item 1)
An isolated nucleic acid molecule comprising:
(A) SEQ ID NO: 1, 2, 3 and 5;
(B) a first polynucleotide encoding a first polypeptide comprising a first amino acid sequence selected from SEQ ID NOs: 7, 8, 9, and 11;
(C) a polynucleotide comprising a nucleotide sequence that is complementary to the first nucleotide sequence of (a); and
(D) a biologically active fragment of (a), (b) or (c),
An isolated nucleic acid molecule comprising a first polynucleotide comprising a first nucleotide sequence selected from:
(Item 2)
The biologically active polypeptide fragment comprises at least 6 consecutive amino acid residues selected from SEQ ID NOs: 7, 8, 9, and 11, wherein at least 2 of the 6 consecutive amino acid residues are Item 8. The nucleic acid molecule according to item 1, which is a leucine residue and arginine residue at amino acid residue positions 80 and 81, respectively.
(Item 3)
2. The nucleic acid molecule according to item 1, wherein the nucleic acid molecule is selected from a cDNA molecule, a genomic DNA molecule, a cRNA molecule, a siRNA molecule, an RNAi molecule, an mRNA molecule, an antisense molecule, and a ribozyme.
(Item 4)
The nucleic acid molecule according to item 1, further comprising its complement.
(Item 5)
2. The nucleic acid molecule of item 1, wherein the first nucleotide sequence is SEQ ID NO: 3.
(Item 6)
2. A nucleic acid molecule that is at least about 70% identical to the nucleic acid molecule of item 1.
(Item 7)
7. The nucleic acid molecule of item 6, wherein said molecule is at least about 80% identical to the nucleic acid molecule of item 1.
(Item 8)
8. The nucleic acid molecule of item 7, wherein said molecule is at least about 90% identical to the nucleic acid molecule of item 1.
(Item 9)
An isolated nucleic acid molecule that specifically hybridizes under stringent conditions to the sequence set forth in SEQ ID NO: 1, 2, or 3, wherein the nucleic acid molecule is a granulocyte, monocyte, and dendritic cell A nucleic acid molecule encoding a polypeptide capable of stimulating proliferation and differentiation.
(Item 10)
An isolated nucleic acid molecule that specifically hybridizes under stringent conditions to the complement of the sequences shown in SEQ ID NOs: 1, 2, 3, and 5, wherein the nucleic acid molecule is granulocyte, monocyte And nucleic acid molecules encoding polypeptides that can stimulate the proliferation and differentiation of dendritic cells.
(Item 11)
6. The nucleic acid molecule according to item 5, further comprising a second sequence.
(Item 12)
12. The nucleic acid molecule of item 11, wherein the second polynucleotide sequence encodes a homologous or heterologous secretory leader.
(Item 13)
13. A nucleic acid molecule according to item 12, wherein the secretory leader is heterologous.
(Item 14)
13. The nucleic acid molecule of item 12, wherein the secretory leader is selected from SEQ ID NOs: 14-211.
(Item 15)
A first amino acid sequence selected from SEQ ID NO: 7, 8, 9 and 11; a sequence selected from the sequences encoded by SEQ ID NO: 1, 2, 3 and 5; and any biologically active thereof An isolated polypeptide comprising a fragment.
(Item 16)
The biologically active fragment comprises at least 6 consecutive amino acid residues selected from SEQ ID NO: 7, 8, 9, and 11, wherein at least 2 of the 6 consecutive amino acid residues are SEQ ID NO: 5. 16. The polypeptide according to item 15, which is leucine and arginine at amino acid residues 80 and 81.
(Item 17)
16. A polypeptide according to item 15, wherein the polypeptide is present in a cell culture.
(Item 18)
16. A polypeptide according to item 15, wherein the polypeptide is present in a cell culture medium.
(Item 19)
18. A polypeptide according to item 17, wherein the cell culture is selected from bacterial cell cultures, mammalian cell cultures, insect cell cultures and yeast cell cultures.
(Item 20)
The polypeptide according to item 15, wherein the polypeptide is present in a plant or a non-human animal.
(Item 21)
16. The polypeptide according to item 15, wherein the first amino acid sequence is the amino acid sequence set forth in SEQ ID NO: 9.
(Item 22)
16. The polypeptide according to item 15, wherein the first amino acid sequence is the amino acid sequence of SEQ ID NO: 7, and the cysteine residue at position 167 is substituted with a serine or alanine residue.
(Item 23)
16. A polypeptide that is at least about 70% and / or at least about 80% homologous to the polypeptide of item 15.
(Item 24)
24. The polypeptide of item 23, wherein the polypeptide is at least about 90% homologous to the polypeptide of item 15.
(Item 25)
An item further comprising a second amino acid sequence, wherein the second amino acid sequence is a homologous secretory leader or a heterologous secretory leader, wherein the first and second amino acid sequences are operably linked. 15. The polypeptide according to 15.
(Item 26)
26. A polypeptide according to item 25, wherein the secretory leader sequence is a heterologous leader sequence.
(Item 27)
27. The polypeptide according to item 26, wherein the heterologous leader sequence is selected from SEQ ID NOs: 14-211.
(Item 28)
A vector comprising the nucleic acid molecule according to item 1 and a promoter that regulates expression of the nucleic acid molecule.
(Item 29)
29. The vector according to item 28, wherein the vector is a viral vector or a plasmid vector.
(Item 30)
29. The vector of item 28, wherein the promoter is selected from a promoter that is naturally adjacent to the nucleic acid molecule and a promoter that is not naturally adjacent to the nucleic acid molecule.
(Item 31)
29. The vector of item 28, wherein the promoter is selected from an inducible promoter, a conditionally activated promoter, a constitutive promoter, and a tissue-specific promoter.
(Item 32)
A recombinant host cell comprising a cell according to item 1 or item 5 and a nucleic acid, a polypeptide according to item 15 or item 25, and / or a vector according to item 28.
(Item 33)
33. A host cell according to item 32, wherein the cell is a prokaryotic cell.
(Item 34)
33. A host cell according to item 32, wherein the cell is a eukaryotic cell.
(Item 35)
35. A host cell according to item 34, wherein the eukaryotic cell is selected from a human cell, a non-human mammalian cell, an insect cell, a fish cell, a plant cell and a fungal cell.
(Item 36)
The host cell of item 32, wherein the cell is a mammalian cell.
(Item 37)
The host cell according to item 36, wherein the mammalian cell is a cell of a 293 cell line or a CHO cell line.
(Item 38)
38. A host cell according to item 37, wherein the cell is a 293 cell.
(Item 39)
40. The host cell according to item 38, wherein the 293 cell is a 293T cell or a 293E cell.
(Item 40)
A non-human animal injected with the nucleic acid molecule according to item 1 or the polypeptide according to item 15.
(Item 41)
41. The animal of item 40, wherein the animal is a rodent, a non-human primate, a rabbit, a dog or a pig.
(Item 42)
6. A nucleic acid composition comprising the nucleic acid molecule according to item 1 or item 5 and a carrier.
(Item 43)
16. A polypeptide composition comprising the polypeptide molecule of item 15 and a carrier.
(Item 44)
29. A vector composition comprising the vector of item 28 and a carrier.
(Item 45)
33. A host cell composition comprising the host cell of item 32 and a carrier.
(Item 46)
43. A composition according to item 42, wherein the carrier is a pharmaceutically acceptable carrier.
(Item 47)
44. The composition of item 43, wherein the carrier is a pharmaceutically acceptable carrier.
(Item 48)
45. A composition according to item 44, wherein the carrier is a pharmaceutically acceptable carrier.
(Item 49)
46. A composition according to item 45, wherein the carrier is a pharmaceutically acceptable carrier.
(Item 50)
A method for producing a recombinant host cell comprising:
(A) providing a vector comprising the nucleic acid molecule of item 1 or item 3; and
(B) contacting the cell with the vector to form a recombinant host cell transfected with the nucleic acid molecule;
Including the method.
(Item 51)
A method for producing a polypeptide comprising:
(A) providing the nucleic acid molecule of item 1 or item 3; and
(B) expressing the nucleic acid molecule in an expression system to produce the polypeptide
Including the method.
(Item 52)
52. The method of item 51, wherein the expression system is a cell expression system.
(Item 53)
53. The method of item 52, wherein the cell expression system is a prokaryotic expression system.
(Item 54)
53. The method of item 52, wherein the cell expression system is a eukaryotic expression system.
(Item 55)
52. The method of item 51, wherein the expression system comprises a host cell transfected with the nucleic acid molecule of item 1 forming a recombinant host cell, and the method comprises culturing the recombinant host cell. And further comprising the step of producing said polypeptide.
(Item 56)
The expression system may be wheat germ lysate, rabbit reticulocyte, ribosome display and E. coli. 52. The method of item 51 which is a cell-free expression system derived from E. coli lysate.
(Item 57)
52. A polypeptide produced by the method of item 51.
(Item 58)
55. A polypeptide produced by the method of item 54, wherein the host cell is selected from mammalian cells, insect cells, plant cells, yeast cells and bacterial cells.
(Item 59)
A diagnostic kit comprising a nucleic acid molecule according to item 1, a reporter for detecting the nucleic acid molecule or its complement, and a vehicle.
(Item 60)
16. A diagnostic kit comprising an antibody that specifically binds to the polypeptide of item 15 and a carrier.
(Item 61)
A diagnostic kit comprising the polypeptide according to item 15 and a carrier.
(Item 62)
A method for determining the presence of an antibody specific for a polypeptide according to item 15 in a patient sample:
(A) providing a composition comprising the polypeptide of item 15;
(B) interacting the polypeptide with the sample;
(C) determining whether an interaction has occurred between the polypeptide and the antibody in the sample, if present;
Including the method.
(Item 63)
An isolated antibody that specifically binds to and / or interferes with the activity of an antigen comprising at least 6 consecutive amino acid residues selected from SEQ ID NOs: 7-12.
(Item 64)
64. The item 63, wherein the consecutive amino acid residues comprise the conserved amino acid residue leu-arg at amino acid positions 80 and 81 of SEQ ID NO: 7, or the consecutive amino acid residue leu-gln-arg of SEQ ID NO: 12. antibody.
(Item 65)
64. The antibody of item 63, wherein the antibody is selected from a polyclonal antibody, a monoclonal antibody, a single chain antibody, and any active fragment thereof.
(Item 66)
Antigen-binding fragment, Fc fragment, cdr fragment, _{V H} fragment is selected from _{V C} fragment and framework fragments, active fragment of claim 65.
(Item 67)
57. The polypeptide of item 15, or the polypeptide produced by the method of any of items 51-56, wherein the polypeptide further comprises at least one fusion partner.
(Item 68)
68. The polypeptide of item 67, wherein the fusion partner is selected from a polymer, polypeptide, succinyl group, fetuin A, fetuin B, leucine zipper domain, tetranectin trimerization domain, mannose binding protein and Fc region.
(Item 69)
69. The polypeptide according to item 68, wherein the polymer is a polyethylene glycol moiety.
(Item 70)
70. The polypeptide according to item 69, wherein the polyethylene glycol moiety is bound via an amino group of an amino acid of the polypeptide.
(Item 71)
70. The polypeptide according to item 69, wherein the polyethylene glycol moiety is branched or a linear polymer.
(Item 72)
A method for screening for a factor that modulates the activity of the polypeptide of item 15, comprising:
(A) providing a test system in which the polypeptide of item 15 affects biological activity; and
(B) screening a plurality of factors for the effect on the activity of the polypeptide of item 15 in the test system;
Including the method.
(Item 73)
73. The method of item 72, wherein the modulator is a small molecule drug.
(Item 74)
73. A method according to item 72, wherein the modulator is an antibody.
(Item 75)
A method for stimulating immune cells comprising:
(A) providing a composition comprising a substantially pure polypeptide selected from any of items 7-12, and an active fragment thereof; and
(B) contacting one or more immune cells with the polypeptide;
Including the method.
(Item 76)
76. The method of item 75, wherein the immune cells are granulocytes, monocytes, lymphocytes, macrophages, peripheral blood mononuclear cells, or dendritic cells.
(Item 77)
77. The method according to item 76, wherein the lymphocyte is an NK cell.
(Item 78)
76. The method of item 75, wherein the polypeptide is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from SEQ ID NOs: 1-6.
(Item 79)
A method for expanding a population of immune cells comprising:
(A) providing a composition comprising a substantially pure polypeptide selected from SEQ ID NOs: 7-12 and any active fragment thereof; and
(B) contacting the polypeptide with one or more immune cells or immune cell precursors;
Including the method.
(Item 80)
80. The method of item 79, wherein the population of immune cells is a population of monocytes, a population of lymphocytes, a population of macrophages, or a population of peripheral blood mononuclear cells.
(Item 81)
81. The method of item 80, wherein the lymphocyte is an NK cell.
(Item 82)
80. The method of item 79, wherein the polypeptide is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from SEQ ID NOs: 1-6.
(Item 83)
A method of stimulating an immune response in a subject comprising:
(A) providing a composition comprising a substantially pure polynucleotide encoding a polypeptide selected from SEQ ID NOs: 7-12 and any active fragment thereof; and
(B) administering the composition to the subject
Including the method.
(Item 84)
84. The method of item 83, wherein the polypeptide is administered locally or systemically.
(Item 85)
85. The method of item 84, wherein said polypeptide is administered intravenously, by enema, intraperitoneally, subcutaneously, topically or transdermally.
(Item 86)
84. The method of item 83, wherein the polypeptide is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from SEQ ID NOs: 1-6.
(Item 87)
A method of increasing immune cells in a subject undergoing cancer treatment comprising:
(A) providing a composition comprising a substantially pure polypeptide selected from any of SEQ ID NOs: 7-12 and any active fragment thereof; and
(B) administering the composition to the subject
Including the method.
(Item 88)
90. The method of item 87, wherein the immune cell is a monocyte, lymphocyte, macrophage, or peripheral blood mononuclear cell.
(Item 89)
90. The method of item 88, wherein the lymphocyte is an NK cell.
(Item 90)
90. The method of item 87, wherein the cancer treatment is selected from chemotherapy and radiation therapy.
(Item 91)
90. The method of item 87, wherein the polypeptide is administered after bone marrow transplantation.
(Item 92)
90. The method of item 87, wherein the polypeptide is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from SEQ ID NOs: 1-6.
(Item 93)
A method of treating or preventing cancer in a subject comprising:
(A) providing a composition comprising a substantially pure polypeptide selected from SEQ ID NOs: 7-12 and any active fragment thereof; and
(B) administering the composition to the subject
Including the method.
(Item 94)
A method of inhibiting tumor growth in a subject comprising:
(A) providing a composition comprising a substantially pure polypeptide selected from SEQ ID NOs: 7-12 and any active fragment thereof; and
(B) administering the composition to the subject
Including the method.
(Item 95)
95. The method of item 94, wherein the tumor comprises human tumor cells.
(Item 96)
96. The method of item 95, wherein the human tumor cells are solid tumor cells or leukemia tumor cells.
(Item 97)
A method of treating or preventing an infection in a subject comprising:
(A) providing a composition comprising a substantially pure polypeptide selected from SEQ ID NOs: 7-12 and any active fragment thereof; and
(B) administering the composition to the subject
Including the method.
(Item 98)
98. The method of item 97, wherein the infection is selected from bacterial infection, mycoplasma infection, fungal infection, viral infection, intracellular pathogen and intracellular parasite.
(Item 99)
98. The method of item 97, wherein the composition is administered locally or systemically to the subject.
(Item 100)
98. The method of item 97, wherein the polypeptide is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from SEQ ID NOs: 1-6.
(Item 101)
A method of modulating an immune response in a subject comprising:
(A) providing a modulator of a polypeptide selected from SEQ ID NOs: 7-12 and any active fragment thereof; and
(B) administering the modulator to the subject
Including the method.
(Item 102)
102. The method of item 101, wherein the modulator is selected from an antibody, a soluble receptor, and a polypeptide.
(Item 103)
103. The method of item 102, wherein the modulator is an antibody.
(Item 104)
102. The method of item 101, wherein the modulator is selected from an aptamer, RNAi, an antisense molecule, and a ribozyme.
(Item 105)
Wherein said antibody is a monoclonal antibody, polyclonal antibody, cdr fragment, V _H fragments, V _C fragment, framework fragments are selected from active fragments of single chain antibodies and antibody The method of claim 103.
(Item 106)
102. The method of item 101, wherein the modulation of immune response is suppression of inflammation.
(Item 107)
102. The method of item 101, wherein the modulation of immune response is suppression of autoimmune disease.
(Item 108)
Item 101. The regulation of the immune response is treatment or prevention of a disease selected from rheumatoid arthritis, osteoarthritis, psoriasis, inflammatory bowel disease, multiple sclerosis, myocardial infarction, stroke and fulminant liver failure. The method described.
(Item 109)
A method of modulating the immune response to pregnancy comprising:
(A) providing a modulator of a polypeptide selected from SEQ ID NOs: 7-12, and any active fragment thereof; and
(B) administering the modulator to a subject
Including the method.
(Item 110)
110. The method of item 109, wherein the modulation of the immune response is a reduction in recurrent miscarriage.
(Item 111)
A method for enhancing an immune response to a vaccine in a subject comprising:
(A) providing a polypeptide composition comprising a substantially pure polypeptide selected from SEQ ID NOs: 7-12 and any active fragment thereof;
(B) providing a vaccine composition; and
(C) A method comprising the step of administering the polypeptide composition and the vaccine composition to the subject.
(Item 112)
111. The method of item 111, wherein the polypeptide composition is administered to the subject prior to administering the vaccine composition.
(Item 113)
111. The method of item 111, wherein the polypeptide composition is administered to the subject after administering the vaccine composition.
(Item 114)
112. The method of item 111, wherein the polypeptide composition is administered to the subject substantially simultaneously with the vaccine composition.
(Item 115)
A method of treating or preventing an inflammatory disease in a subject comprising:
(A) providing a modulator of a polypeptide selected from SEQ ID NOs: 7-12 and any active fragment thereof; and
(B) administering the modulator to the subject
Including the method.
(Item 116)
116. The method of item 115, wherein the modulator is selected from aptamers, RNAi, antisense molecules, and ribozymes.
(Item 117)
116. The method of item 115, wherein the modulator is selected from an antibody, a soluble receptor, and a polypeptide.
(Item 118)
118. The method according to item 117, wherein the modulator is an antibody.
(Item 119)
Wherein said antibody is a monoclonal antibody, polyclonal antibody, cdr fragment, V _H fragments, V _C fragment, framework fragments are selected from active fragments of single chain antibodies and antibody The method of claim 118.
(Item 120)
A method of treating or preventing an autoimmune disease in a subject comprising:
(A) providing a modulator of a polypeptide selected from SEQ ID NOs: 7-12, and any active fragment thereof; and
(B) administering the modulator to the subject
Including the method.
(Item 121)
121. The method of item 120, wherein the modulator is selected from an antibody, aptamer, soluble receptor, RNAi molecule, antisense molecule, ribozyme and polypeptide.
(Item 122)
Wherein said antibody is a monoclonal antibody, polyclonal antibody, cdr fragment, V _H fragments, V _C fragment, framework fragments are selected from active fragments of single chain antibodies or antibody The method of claim 121.
(Item 123)
A method for increasing the number of NK cells in a subject comprising:
(A) providing a polypeptide selected from SEQ ID NOs: 7-12, and any active fragment thereof; and
(B) administering the polypeptide to the subject
Including the method.
(Item 124)
A method of modulating an NK cell population in a subject comprising:
(A) providing a polypeptide selected from SEQ ID NOs: 7-12, and any active fragment thereof; and
(B) administering the polypeptide to the subject
Including the method.
(Item 125)
125. A method according to item 124, wherein the modulator is selected from an antibody, aptamer, soluble receptor, RNAi molecule, antisense molecule, ribozyme and polypeptide.
(Item 126)
Wherein said antibody is a monoclonal antibody, polyclonal antibody, cdr fragment, V _H fragments, V _C fragment, framework fragments are selected from active fragments of single chain antibodies and antibody The method of claim 125.
(Item 127)
126. The method of item 124, wherein the NK cell population stimulates an immune response.
(Item 128)
125. A method according to item 124, wherein the NK cell population suppresses miscarriage.
(Item 129)
125. The method of item 124, wherein the NK cell population stimulates an anti-cancer response.
(Item 130)
A method for expanding a population of hematopoietic stem cells comprising:
(A) providing a composition comprising a substantially pure polypeptide selected from SEQ ID NOs: 7-12, and any active fragment thereof;
(B) contacting the polypeptide with the population of hematopoietic stem cells
Including the method.
(Item 131)
A method for providing a cytoprotective effect on a population of cells comprising:
(A) providing a composition comprising a substantially pure polypeptide selected from SEQ ID NOs: 7-12, and any active fragment thereof;
(B) contacting the population of cells with the polypeptide
Including the method.
(Item 132)
134. The method of any of items 101, 109, 111, 115, 123, 124, 130, or 131, wherein the composition is administered locally or systemically to the subject.
(Item 133)
Items 101, 109, 111, 115, 120, 123, 124, 130, wherein said polypeptide is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from SEQ ID NOs: 1-6 and any active fragment thereof. Or the method according to any one of 131.
(Item 134)
132. The method according to any of items 101, 109, 111, 115, 120, 123, 124, 130 or 131, wherein said polypeptide further comprises at least one fusion partner.
(Item 135)
135. A method according to item 134, wherein the fusion partner is selected from a polymer, polypeptide, fetuin and serum albumin.
(Item 136)
135. A method according to item 134, further comprising a polymer and / or a succinyl group.
(Item 137)
139. The method of item 136, wherein the polymer is a polyethylene glycol moiety.
(Item 138)
138. The method of item 137, wherein the polyethylene glycol moiety is attached via the amino group of an amino acid of the polypeptide.
(Item 139)
140. The method of item 137, wherein the polyethylene glycol moiety is a branched polymer or a linear polymer.

FIG. 1 shows exons of MGD-CSF (MGD-CSF_exon 4); SEQ ID NO: 8), MGC34647 (NP_686969); SEQ ID NO: 10) and MGD-CSF SEQ ID NO: 7) as further described in Example 1. An amino acid sequence alignment of 4 is shown. Amino acid identity is designated by (*). FIG. 2 shows a diagram of the pTT5 backbone vector used to generate pTT5-Gateway used to transiently transfect mammalian cells, as further described in Example 2. FIG. 2 also shows pTT5-G (residues 1182-1265 and 2984-3127 of SEQ ID NO: 272), pTT5-H (residues 1182-1392 and 1597-1602 of SEQ ID NO: 273) and pTT5-I (SEQ ID NO: 284). And the nucleic acid sequence of the multicloning site adjacent to the vector sequence of residues 2905-3048) of SEQ ID NO: 274. FIG. 2 shows transient transformation of mammalian cells, as further described in Example 2. A diagram of the pTT5 backbone vector used to generate the pTT5-Gateway used to transfect is shown. FIG. 2 also shows pTT5-G (residues 1182-1265 and 2984-3127 of SEQ ID NO: 272), pTT5-H (residues 1182-1392 and 1597-1602 of SEQ ID NO: 273) and pTT5-I (SEQ ID NO: 284). And the nucleic acid sequence of the multicloning site adjacent to the vector sequence of residues 2905-3048) of SEQ ID NO: 274. FIG. 3 shows a diagram of the pTT2 backbone vector used to stably transfect mammalian cells, as further described in Example 2. FIG. 4 shows the expression of MGD-CSF in plasmid vectors 3-6 days after transfection, as further described in Example 3 and Table 1. FIG. 4A shows the expression of MGD-CSF vector with C-terminal His tag, intracellular (cell) and secreted (supernatant) CLN00732663 in 293-6E cells. FIG. 4B shows expression of MGD-CSF vector with intracellular C-terminal His tag and collagen leader sequence, intracellular (cell) and secreted (supernatant) CLN00816424 in 293-6E cells. Both FIG. 4A and FIG. 4B show Positope ^™ (Invitrogen, Carlsbad, Calif.) (Right panel) as a positive control for expression. Molecular weights are shown on the left panel. FIG. 5 shows the extent of growth and viability of cells transfected with the constructs described in Example 2 and Table 1, 3-6 days after transfection, as further described in Example 4 and Table 1. Indicates. FIG. 5A shows cells transfected with CLN00542945 (black bars), CLN00732663 (light gray bars), CLN00821867 (hatched bars), and CLN00816424 (lattice bars), and secreted alkaline phosphatase (SEAP). The degree of cell proliferation compared to the encoded control gene (dark gray bar) is shown. Cells transfected with both CLN00816424 and control SEAP increased in cell number 3-6 days after transfection, as further described in Example 4. FIG. 5B is transfected with CLN00542945 (black bars), CLN00732663 (light gray bars), CLN00821867 (shaded bars), and CLN00816424 (lattice bars) to encode secreted alkaline phosphatase (SEAP). The percentage of cell viability compared to cells transfected with the control gene (dark gray bar) is shown. Cells transfected with CLN00816424 and control SEAP cells remained alive, whereas cells transfected with MGD-CSF, CLN00732663 and CLN00821867 showed increased toxicity, depending on the culture conditions, It was not gene specific but was evidenced by their reduced viability over time in culture. FIG. 6 shows that adherent 293-T cells expressing MGD-CSF can be adapted to low serum concentration culture conditions, as further described in Example 5. FIG. 6A is in suspension 293-T cell culture grown in FreeStyle medium with 3% FBS and in HyQ-CHO medium with 1% FBS, as further described in Example 5. Shows the expression of MGD-CSF. FIG. 6B shows low serum (panel 1) in the absence of serum (panel 2) in suspension culture and adherent culture compared to purified MGD-CSF standards generated from bacterial hosts (panel 4). In (Panel 3), the expression of MGD-CSF is shown. FIG. 7 shows the expression of MGD-CSF in a bioreactor as further described in Example 6. The fermentation was monitored for 6 days. Samples were examined by gel electrophoresis and stained with Coomassie blue stain 1-6 days after inoculation. Molecular weights are shown in the left panel. The position of MGD-CSF is indicated by an arrow. In order to quantify the amount of protein on the gel, increasing amounts of bovine serum albumin (BSA) are shown. FIG. 8A shows the isolation of MGD-CSF from 293-T cells, as further described in Example 7. Cell culture supernatant (Supt) was fractionated into Sp-Sepharose FF column (SP-Pool), Heparin Sepharose HP column (Hep-Pool), and Q-Sepharose column (Q-Pool). MFD-CSF is glycosylated and has an apparent molecular weight of 39 kDa by SDS-polyacrylamide gel electrophoresis. Molecular weight markers are shown in the left panel. FIG. 8B shows the electrophoretic migration pattern of MGD-CSF mutein (mutein) under reducing and non-reducing SDS-PAFE conditions, as described in more detail in Example 8. The left panel shows an SDS-PAGE gel run under conditions that reduce disulfide bonds, and the middle panel shows an SDS-PAGE gel run under conditions that do not reduce disulfide bonds. Wild-type MGD-CSF, wild-type MGD-CSF expressed with its natural signal peptide (wt natSP), wild-type MGD-CSF, wild-type expressed with collagen signal peptide (wt colSP) Mutation of MGD-CSF and the cysteine to serine mutein (mutein) C35S, C167S, C176S, C178S, C179S, C190S and C198S are shown in both panels. Purified MGD-CSF is shown in the right panel as a standard for quantification and comparison. C179S and C190S were expressed at lower levels than wild type MGD-CSF, and C35S was not expressed at detectable levels. FIG. 9 shows the ability of MGD-CSF to induce NK cell proliferation and / or survival, as further described in Example 9A. The number of NK cells was expressed as relative luciferase units (RLU) per well after exposure to conditioned medium (squares) from cells transfected with negative buffer control (diamonds) or MGD-CSF. MGD-CSF stimulated mouse NK cell proliferation. Control buffer had no effect. The proliferative activity of MGD-CSF against NK cells was specific and dose dependent. FIG. 10 shows the results of a screening assay for factors that induce NK cell proliferation and / or survival, as further described in Example 9A. The upper and lower panels show two identical experiments performed independently. The number of human NK cells is expressed in relative luciferase units (rlu) after exposure to the test agent. Conditioned media from cells transfected with MGD-SCF or with plasmid DNA from cluster 190647, the source of MGD-CSF, stimulated the survival and / or proliferation of human NK cells. Positive controls IFNγ and GM-CSF also stimulated NK cell proliferation. FIG. 11 shows the ability of MGD-CSF to induce hematopoietic stem cell proliferation, as further described in FIG. 9B. The number of stem cells was determined by counting the cells with a hemocytometer. MGD-CSF increased proliferation in a dose-dependent manner. MGD-CSF (500 ng / ml) induced stem cell proliferation to a greater extent than M-CSF and to a similar extent to G-CSF and GM-CSF. FIG. 12 shows the ability of MGD-CSF to induce myeloid cell proliferation, as further described in Example 9C. FIG. 12A shows the ability of conditioned medium containing MGD-CSF to induce bone marrow proliferation with negative control empty vector (Vector) and irrelevant compounds CLN3732, FPT026, IL-10 and non-conditioned medium; Compare with control GM-CSF. The number of monocytes was expressed as relative luciferase units (RLU) per well after exposure to the test agent. FIG. 12 shows the ability of MGD-CSF to induce myeloid cell proliferation, as further described in Example 9C. FIG. 12B shows that both conditioned media from cells transfected with purified GM-CSF (stars) and MGD-CSF (squares) stimulated human monocyte proliferation. The control vector (diamond) had no effect. The proliferative activity of MGD-CSF on monocytes was specific and dose dependent. FIG. 13 shows the results of a fluorescence activated cell sorting (FACS) analysis of granulocyte differentiation as measured in the presence of differentiation antigens CD67 and CD24 and as described in more detail in Example 10A. Show. The number of hematopoietic stem cells induced to differentiate into granulocytes in response to a medium conditioned with a negative control vector (Vector CM) and a medium conditioned with an MGD-CSF vector (MGD-CSF CM) is shown. CD67 FITC (x axis) indicates the number of CD67 positive cells according to the fluorescence intensity of an antibody specific for CD67. CD24PE (y-axis) indicates the number of CD24 positive cells by the fluorescence intensity of an antibody specific for CD24. The triangular area (arrow) surrounded by a line indicates the number of cells positive for both cell surface differentiation antigens CD67 and CD24 in each of the four panels. MGD-CSF CM stimulated granulocyte differentiation both in the absence (no cytokine) and presence of G-CSF. This stimulation by MGD-CSF was synergistic with the effect of G-CSF. FIG. 14 shows the results of FACS analysis of granulocyte differentiation as measured by the presence of differentiation antigens CD15 and CD24 and as described in further detail in Example 10A. CD24PE (x axis) indicates the number of CD24 positive cells according to the fluorescence intensity of an antibody specific for CD24. CD15APC (y-axis) indicates the number of CD15 positive cells by the fluorescence intensity of an antibody specific for CD15. The right-hand pane above each graph shows the percentage of those cells that have both CD15 and CD34 differentiation markers on the cell surface. MGD-CSF induced granulocyte differentiation in a concentration-dependent manner, where a dose of 500 ng / ml resulted in differentiation of 8% of bone marrow hematopoietic stem cells into granulocytes. FIG. 15 shows the results of FACS analysis of monocyte differentiation as measured by the presence of differentiation antigens CD14 and CD3 and as described in further detail in Example 10B. The number of hematopoietic stem cells induced to differentiate into monocytes in response to media conditioned with a negative control vector (Vector CM) and media conditioned with MGD-CSF vector (MGD-CSF CM) is shown. CD14FITC (x-axis) indicates the number of CD14-positive cells according to the fluorescence intensity of an antibody specific for CD14. CD3APC (y-axis) indicates the number of CD3 positive cells by the fluorescence intensity of an antibody specific for CD3. Lined oval regions (arrows) indicate the number of cells positive for both cell surface differentiation antigens CD14 and CD3 in each of the four panels. MGD-CSF CM stimulated monocyte differentiation. MGD-CSF CM stimulated monocyte differentiation both in the absence (no cytokine) and presence of G-CSF. The ability of MGD-CSF to induce monocyte differentiation was greater than that of GM-CSF. MGD-CSF acted synergistically with GM-CSF. FIG. 16 shows the results of FACS analysis of monocyte differentiation as measured by the presence of differentiation antigens CD14 and CD16 and as described in further detail in Example 10B. CD14APC (y-axis) indicates the number of CD14-positive cells by the fluorescence intensity of an antibody specific for CD14. CD16FITC (x axis) indicates the number of CD16 positive cells according to the fluorescence intensity of an antibody specific for CD16. The right-hand pane above each graph shows the percentage of those cells that have both CD14 and CD16 differentiation markers on the cell surface. MGD-CSF induced monocyte differentiation in a concentration-dependent manner, where a dose of 100 ng / ml resulted in differentiation of 10% of bone marrow hematopoietic stem cells into granulocytes. FIG. 17 shows the results of FACS analysis of dendritic cell differentiation as measured by the presence of differentiation antigens CD86 and CD1 and as described in further detail in Example 10C. The number of hematopoietic stem cells induced to differentiate into dendritic cells in response to media conditioned with a negative control vector (Vector CM) and media conditioned with MGD-CSF vector (MGD-CSF) is shown. CD1aFITC (x axis) indicates the number of CD1-positive cells according to the fluorescence intensity of an antibody specific for CD1. CD86PE (y-axis) indicates the number of CD86-positive cells by the fluorescence intensity of an antibody specific for CD86. The oval region surrounded by a line indicates the number of cells positive for both cell surface differentiation antigens CD1 and CD86. MGD-CSF CM stimulated dendritic cell differentiation. Of the cells transfected with the negative control, 4% differentiated into dendritic cells, whereas 22% of the cells transfected with the MGD-CSF vector differentiated into dendritic cells. FIG. 18 shows the effect of MGD-CSF on human bone marrow colony formation, as described in more detail in Example 11. FIG. 18A shows the dose-dependent stimulatory effect of MGD-CSF on the formation of CFU-G (left panel) and CFU-M (right panel) compared to the effects of G-CSF and GM-CSF. FIG. 18 shows the effect of MGD-CSF on human bone marrow colony formation, as described in more detail in Example 11. FIG. 18B shows the dose-dependent stimulation of MGD-CSF on CFU-GM formation (left panel) and total colony forming ability (Total CFC) (right panel) compared to the effects of G-CSF and GM-CSF. Show the effect. FIG. 18 shows the effect of MGD-CSF on human bone marrow colony formation, as described in more detail in Example 11. FIG. 18C shows CFU-G (upper left panel), CFU-GM (upper right panel) compared to the effects of G-CSF and GM-CSF in the presence of cytokine IL-3 and stem cell factor (SCF). ), Shows the dose-dependent stimulatory effect of MGD-CSF on the formation of CFU-M (lower left panel) and total colony forming ability (Total CFC) (lower right panel). FIG. 19 shows a profile of the effect of MGD-CSF in assays for various biological activities, as described in more detail in Example 12. MGD-CSF stimulated proliferation of activated monocytes (MonPro4) and peripheral NK cells (NKGlo). FIG. 20 shows the profile of the effect of MGD-CSF in various assays of cytokine secretion, as described in more detail in Example 13. MGD-CSF stimulated the secretion of GM-CSF (Luminex2-GMCSF), IL-2 (Luminex2-IL2) and IL-13 (Luminex2-IL13). FIG. 21 shows the effect of MGD-CSF on CFU-M formation, as described in more detail in Example 14. Examples of CFU-M formation induced by buffer, GM-CSF and G-CSF are shown in the top three panels. The dose-dependent effects of CFU-M formation induced by 20 ng / ml, 100 ng / ml and 500 ng / ml MGD-CSF are shown in the lower three panels. FIG. 22 shows the effect of MGD-CSF on dendritic cell formation, as described in more detail in Example 15. Examples of dendritic cell formation induced by media, 20 ng / ml MGD-CSF, 100 ng / ml MGD-CSF and 500 ng / ml MGD-CSF are shown. MGD-CSF induced the formation of elongated differentiated dendritic cells from spherical undifferentiated hematopoietic stem cells.

(Description of Embodiment)
(Definition)
Terms used herein have their ordinary meanings as indicated below and can be further understood in the context of this specification.

  “Monocyte, granulocyte and dendritic cell colony stimulating factor” (MGD-CSF) is a novel, isolated, secreted molecule comprising: Molecules having nucleic acid and amino acid sequences as shown in SEQ ID NOs: 1 and 7, respectively. Provisional applications 60 / 590,565 and 60 / 564,932 called MGD-CSF FPT025. The term “molecules of the invention” refers to any of SEQ ID NOs: 1-13, secretory leaders of any of SEQ ID NOs: 1-13 and SEQ ID NOs: 14-211, and As used herein as comprising any of the ~ 271 constructs.

  The terms “nucleic acid molecule”, “nucleotide”, “polynucleotide” and “nucleic acid” are intended to refer to nucleotides of any length in polymer form. Used interchangeably in the specification. They may include both double-stranded and single-stranded sequences and include, but are not limited to, cDNAs from viral, prokaryotic and eukaryotic sources; mRNA; viruses (eg, DNA viruses and retroviruses). Virus) or genomic DNA sequences from prokaryotic sources; RNAi; cDNA; antisense molecules; ribozymes; and synthetic DNA sequences. The term also encompasses sequences that include any known base analog of DNA and RNA.

  “Recombinant” as used herein refers to a nucleic acid molecule, a polynucleotide derived from a genome, cDNA, virus, semi-synthetic and / or synthetic, thanks to its origin or manipulation. By a polynucleotide it is not associated with all or part of the polynucleotide with which it is naturally associated. The term “recombinant” when used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. The term “recombinant” when used with respect to a host cell means a host cell into which a recombinant polynucleotide has been introduced.

  A “complementary” nucleotide sequence nucleic acid molecule consists of the complement of its base pair. Deoxyribonucleotides with base adenine are complementary to deoxyribonucleotides with base thymidine, and deoxyribonucleotides with base thymidine are complementary to deoxyribonucleotides with base adenine. A deoxyribonucleotide having a base cytosine is complementary to a deoxyribonucleotide having a base guanine, and a deoxyribonucleotide having a base guanine is complementary to a deoxyribonucleotide having a base cytosine. Ribonucleotides with base adenine are complementary to ribonucleotides with base uracil, and ribonucleotides with base uracil are complementary to ribonucleotides with base adenine. Ribonucleotides with the base cytosine are complementary to ribonucleotides with the base guanine, and deoxyribonucleotides with the base guanine are complementary to deoxyribonucleotides with the base cytosine.

  A “promoter” as used herein is a DNA capable of binding RNA polymerase in a mammalian cell and initiating transcription of a downstream (3 ′ direction) coding sequence operably linked thereto. It is a regulatory region. For the purposes of the present invention, the promoter sequence contains the minimum number of bases or elements necessary to initiate transcription of the gene of interest at a detectable level above background. The promoter sequence may be within the transcription initiation site and may also be a protein binding domain (consensus sequence) responsible for binding of RNA polymerase. Eukaryotic promoters, but not always, often include a “TATA” box and a “CAT” box. Promoters include promoters that are naturally close to nucleic acid molecules and promoters that are not naturally close to nucleic acid molecules. Furthermore, the term “promoter” includes inducible promoters, conditionally active promoters such as the cre-lox promoter, constitutive promoters and tissue specific promoters.

  “Operatively linked” refers to an arrangement of elements in which the components so represented are configured to perform their desired function. Thus, a secretory leader sequence operably linked to a polypeptide sequence can achieve secretion of the polypeptide from the cell.

  “Transfected” means to retain the introduced DNA or RNA with or without the use of any co-promoter such as lipofectamine. Methods of transfection known in the art include calcium phosphate transfection, DEAE dextran transfection, protoplast fusion, electroporation and lipofection.

  “Expression of nucleic acid molecule” refers to the conversion of information contained in this nucleic acid molecule into a gene product. A gene product is a direct transcript of a gene (eg, mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a peptide or polypeptide produced by translation of mRNA It can be. Gene products also include RNA modified by processes such as capping, polyadenylation, methylation and editing; and, for example, by methylation, acetylation, phosphorylation, ubiquitin binding, ADP ribosylation, myristoylation and glycosylation Examples include proteins to be modified.

  A “vector” is an agent, typically a virus or a plasmid, that can be used to convert genetic material into a cell or organism.

  A “host cell” is an individual that can be or has been a recipient of any recombinant vector (s) or isolated polynucleotide (s). A cell or cell culture. A host cell contains the progeny of a single host cell, and this progeny is completely identical to the original parent cell due to natural, accidental or intentional mutations and / or changes. There is no need (morphologically or in the total DNA complement). Host cells include cells that have been transfected or infected in vivo or in vitro using the recombinant vectors or polynucleotides of the present invention. A host cell containing a recombinant vector of the present invention may be referred to as a “recombinant host cell”.

  A “stem cell” is an undifferentiated pluripotent or multipotent cell that self-regenerates, remains undifferentiated, and has the ability to differentiate. Stem cells can divide, but are not limited to, at least for the life of the animal in which the stem cells are naturally present. Stem cells are not terminally differentiated, which means that they are not the end of the differentiation pathway. When the stem cells divide, each daughter cell may remain in the stem cell, or the daughter cell may embark on a pathway that results in terminal differentiation. The stem cell may be an embryonic stem cell, a juvenile stem cell, or an adult stem cell. “Hematopoietic stem cells” are involved in the process of hematopoiesis, a process of forming mature blood cells from progenitor cells.

  The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Thus, peptides, oligopeptides, dimers, multimers, etc. are included within this definition. Both full-length proteins and fragments thereof are encompassed by this definition. The term also encompasses post-expression modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, and the like. Further, for purposes of the present invention, a “polypeptide” is a deletion, addition, and substitution (generally conservative in nature) relative to the native sequence, so long as the protein maintains the desired activity. A protein containing such modifications. These modifications may be intended, such as through site-directed mutagenesis, or may be incidental, such as through mutations in the host producing the protein, or errors due to PCR amplification.

  A “leader sequence” is a sequence of amino acid residues that begins at amino acid residue 1 located at the amino terminus of a polypeptide and extends to a cleavage site that results in the formation of a mature protein upon protein cleavage. including. The leader sequence is generally hydrophobic and has a number of positively charged residues. Leader sequences may be natural or synthetic, heterologous to the protein to which they are bound, or homologous. A “secretory leader” is a leader sequence that indicates that a protein is secreted from a cell.

  A “fusion partner” is fused in-frame at the N-terminus and / or C-terminus of a therapeutic or prophylactic polypeptide, or internally to a therapeutic or prophylactic polypeptide It is a polypeptide.

  The term “receptor” refers to a polypeptide that binds to a specific ligand. The ligand is usually an extracellular molecule that, upon binding to the receptor, usually initiates a cellular response, such as the initiation of a signaling pathway. A “soluble receptor” is a receptor that lacks a membrane-bound domain, such as a transmembrane domain. “Soluble receptors” may include naturally occurring splicing variants of wild-type transmembrane protein receptors in which the transmembrane domain has been unspliced. A “soluble receptor” may include the extracellular domain of a transmembrane protein receptor or any fragment of the extracellular domain. Soluble receptors can modulate the target protein. They may, for example, compete with wild-type receptors for ligand binding and may be involved in ligand / receptor interactions, whereby the activity or number of receptors and / or downstream from this receptor Modulates cellular activity. This modulation may trigger an intracellular response, such as a signaling event that activates the cell, a signaling event that inhibits the cell, or an event that regulates cell growth, proliferation, differentiation and / or death, or Induces the production of other factors that in turn regulate such activities.

  An “isolated”, “purified”, “substantially isolated” or “substantially pure” molecule (eg, a polypeptide Or a polynucleotide) is a molecule that has been engineered to be present at a higher concentration than in its natural state. For example, an antibody of the invention can be used when at least 10% or 20% or 40% or 50% or 70% or 90% of the non-antibody material with which it is naturally associated has been removed. Isolated, purified, substantially isolated, or substantially purified. As used herein, an “isolated”, “purified”, “substantially isolated” or “substantially purified” molecule includes a recombinant molecule.

  An entity that is “biologically active” or that has “biological activity” is a structural, regulatory or biochemical function of a naturally occurring molecule, or An entity having any function related to or related to a metabolic or physiological process. Biologically active polynucleotide fragments are fragments that exhibit similar, but not necessarily identical, activity to the polynucleotides of the invention. This biological activity may include an improved desired activity or a reduced undesirable activity. For example, when an entity is involved in a molecular interaction with another molecule, eg, has a therapeutic role in alleviating hybridization, a disease state, it has a prophylactic value in inducing an immune response. In determining the presence of a molecule such as a biologically active fragment of a polynucleotide that can be determined to be unique for a polynucleotide molecule or can be used as a primer in a polymerase chain reaction A biological activity is indicated if it has a therapeutic and / or prophylactic value. Biologically active polypeptides or fragments thereof include those that can participate in a biological response, including but not limited to stimulating an immune response such as the production of antibodies. Those that can function as epitopes or immunogens; or those that can be involved in modulating the immune response.

The terms “antibody” and “immunoglobulin” are used interchangeably to refer to a protein, eg, a protein that is produced synthetically or recombinantly by the immune system and that is a specific antigen. Refers to a protein that can recognize and bind to it. Antibodies are generally known in the art. The antibody can recognize a polypeptide or polynucleotide antigen. The term encompasses active fragments, including, for example, immunoglobulin antigen-binding fragments, heavy chain variable and / or constant regions, light chain variable and / or constant regions, complementarity determining regions (cdr). ), And framework areas. The term includes polyclonal and monoclonal antibody preparations, as well as hybrid antibodies, altered antibodies, chimeric antibodies, hybrid antibody molecules, F (ab ′) ₂ and F (ab) fragments; Dimer), dimer and trimer antibody fragment constructs; minibodies, humanized antibody molecules and any functional fragment derived from such molecules, wherein such fragments are specific Fragments that retain general binding are included.

  A “vaccine” is a preparation that immunizes or artificially augments a particular disease. This is done from compounds that give rise to immunity to a specific disease or to be artificially augmented, for example from killed microorganisms, live attenuated organisms, or live pathogenic organisms Can be configured. This includes preparations that contain a type of attenuated or killed microorganism that produces a particular disease that is administered to stimulate the immune system to produce antibodies against that disease. The term includes nucleic acid and polypeptide vaccines.

  The term “binds specifically” refers to high binding activity and / or high affinity binding to a particular epitope in the context of antibody binding. Thus, an antibody that specifically binds to one epitope (a “first epitope” and not another (“second epitope”)) An antibody specific for a first epitope may cross-react with and bind to a second epitope if the two epitopes share homology or other similarities. The term “specifically binds”, in the context of polynucleotides, refers to hybridization under stringent conditions, increasing the stringency of both DNA / DNA and DNA / RNA hybridization reactions. Conditions are widely known and published in the art (Curr. Prot. olec.Biol., John Wiley & Sons (2001)).

  The terms “subject”, “individual”, “host” and “patient” are used interchangeably herein and include human and non-human animals. A live animal. A subject may be, for example, an organism carrying immune cells that can respond to antigenic stimulation and stimulatory and inhibitory signaling through cell surface receptor binding. The subject may be a mammal, such as a human or non-human mammal, such as a dog, cat, pig, cow, sheep, goat, horse, rat and mouse. The term “subject” does not exclude individuals who are completely normal with respect to the disease or are normal in all respects.

  A “patient sample” is any biological specimen from a patient. The term includes, but is not limited to, biological fluids such as blood, serum, plasma, urine, cerebrospinal fluid, tears, saliva, lymph, dialysate, lavage fluid, semen and other fluid samples, and biological Includes cells and tissues of scientific origin. The term also includes cells and cells derived therefrom and their progeny, including cells in culture, cell supernatants and cell lysates. This was further separated from fluids derived from organ or tissue culture, tissue biopsy samples, tumor biopsy samples, stool samples and biological tissue, and solid tissues, tissue sections and cell lysates Includes cells. This definition encompasses samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization or enrichment for certain components, such as polynucleotides or polypeptides. The term also includes patient sample derivatives and fractions. Patient samples can be used in diagnostic, prognostic or other monitoring assays.

  A “disease” is a pathological condition, eg, a pathological condition that can be identified by symptoms or other identifying factors as distinct from a healthy or normal condition. The term “disease” encompasses disorders, syndromes, conditions and injuries. Diseases include, but are not limited to, proliferative, inflammatory, immune, metabolic, infectious, and ischemic diseases.

  The term “modulate” refers to the production of either an increase or decrease, stimulation, inhibition, interference, or interference, either directly or indirectly, in measured activity when compared to an appropriate control. Say. Polypeptide or polynucleotide, or “modulator” of an “agent”, is used interchangeably herein to refer to a polypeptide or polynucleotide when compared to an appropriate control. A term that refers to a substance that affects, for example, increases, decreases, stimulates, inhibits, interferes with or blocks measured activity.

  “Preventing” as used herein refers to providing prophylaxis for the onset or recurrence of a disease in a subject who may be predisposed to the disease but has not yet been diagnosed with the disease. Includes. Treatment and prevention can be administered in vivo, in vitro or ex vivo to an organism, including a human, or to a cell, and the cell can subsequently be administered to the subject.

  “Treatment” as used herein includes any administration or application of a therapeutic agent for a disease in a mammal, including a human, and includes inhibiting the disease. This includes preventing the progression of the disease, such as by suppressing or restoring or repairing loss of function, deletion or deficiency, or stimulating inadequate processes, and relieving the disease To do.

  “Carrier” refers to a solid, semi-solid or liquid filler, diluent, encapsulating agent, formulation aid, or any conventional type of excipient. “Pharmaceutically acceptable carrier” refers to a non-toxic “carrier”. Pharmaceutically acceptable carriers are non-toxic to recipients at the dosages and concentrations used and are compatible with the other ingredients of the formulation. A pharmaceutically acceptable carrier can be, for example, a vehicle, an adjuvant or a diluent.

(MGD-CSF and related nucleic acids and polypeptides)
The present invention is a novel isolated secreted molecule comprising "monocytes, granulocytes and dendritic cell colony stimulating factor (monocyte, granulocyte, and dendritic).
A secreted molecule identified herein as "cell colony stimulating factor" (MGD-CSF) is provided. The present invention provides methods using MGD-CSF, and related factors including MGD-CSF variants and variants. MGD-CSF is related to NP_686969, Molecular Genomics Clone MGC34647, and Incyte SEQ ID NOs: 232, 255, and 257 (WO 2002/048337), as described further below.

  MGD-CSF is 241 amino acids long and contains a signal peptide or secretory leader sequence. MGD-CSF is a subclone derived from the mother clone CLN00506579 in the clone family CLN00212388. MGD-CSF belongs to Five Primer cluster 190647. This cluster of secreted proteins contains all the expressed sequences corresponding to a single gene. The situation is confirmed by its Trevote of 0.92 as a secreted molecule. This Threevote is the result of an algorithm built on a number of physical and chemical attributes that predict whether the predicted amino acid sequence will be secreted; a Treevote greater than 0.50 is a secreted molecule It is an indicator.

As shown in FIG. 1, MGD-CSF is National Center for.
It relates to a hypothetical protein that is predicted to be encoded by the mRNA sequence of NP — 688669, named by Biotechnology (NCBI) (Strausberg et al., Proc. Natl. Acad. Sci. 99: 16,899 (2002)). This hypothetical human protein is predicted to contain 242 amino acids. The coding sequence of NP_689669 is described as MCG34647 by the National Institute of Health, National Gene Collection (MGC). The nucleic acid sequence of MGC34647 corresponds to SEQ ID NO: 49 and SEQ ID NO: 103, respectively, as specified in WO 2002/048337, where SEQ ID NO: 49 is a secreted protein of unknown function encoded by SEQ ID NO: 103 As described. The functions of MGC34647 and NP_6869669 have not been disclosed in the past. MGD-CSF is a novel splicing variant of MGC34647. The junction between exon 3 and exon 4 is differentially spliced such that amino acid L80 is followed by R81.

  The gene MGC34647 is predicted to encode a protein with a 242 amino acid open reading frame having a nucleic acid coding sequence of 729 nucleotides in length. This precursor protein is weighed to 27,479 daltons and is expected to have an isoelectric point of 7.72. After cleavage of the signal peptide containing amino acids 1-20, this mature protein is weighed to 25,229 daltons and is expected to have an isoelectric point of 6.74. This mature protein is predicted to be 222 amino acids long, encoded by a 669 nucleotide nucleic acid molecule. MGC34647 has six exons. This maps to the genome on chromosome 16q22.1 from the start position of 704656649 to the stop position of 70470765.

  MGC34647 expression has been observed in the spleen, parotid gland, meniscus of joints, bile ducts, seminal vesicles, medulla, pituitary gland, salivary glands and sequence listing, as further described below. Based on these localizations, MGC34647 is expected to have several specific therapeutic uses. This may be the target of antagonistic antibodies for autoimmune diseases such as multiple sclerosis, rheumatoid arthritis and systemic lupus erythematosus (SLE). This can be, for example, as a protein therapeutic agonist for hematopoietic cell regeneration during chemotherapy and bone marrow transplantation, as an antagonistic protein therapeutic to enhance cell-mediated immunity to treat infectious diseases, or It can be used as a protein therapeutic antagonist for cell protection.

(Nucleic acid)
The present invention provides nucleic acid molecules comprising polynucleotide sequences corresponding to the novel MGD-CSF sequences as set forth in the Tables and Sequence Listing, eg, SEQ ID NOs: 1, 2, 3, and 5. The present invention provides uses for these nucleic acid molecules and for related nucleic acid molecules, eg, the nucleic acid molecules shown in SEQ ID NOs: 4 and 13. These uses are described herein.

  The present invention relates to a liver-expressed gene promoter operably linked to a gene encoding MGD-CSF or NP-688669, and a DNA molecule that can be expressed in vivo to yield a functionally active protein. I will provide a. The described DNA molecules can be used in a variety of applications, for example, in vivo function of artificially introduced MGD-CSF or NP_688669, interaction of two or more artificially introduced MGD-CSF or NP_688669, artificial As a tool in basic research to study the in vivo kinetics of MGD-CSF or NP_688669 fusion proteins introduced into or to identify in vivo targets of artificially introduced MGD-CSF or NP_688669 proteins, and below As a therapeutic treatment as further described in, it has various uses.

  Non-limiting embodiments of nucleic acid molecules include genes or gene fragments, exons, introns, mRNA, tRNA, rRNA, siRNA, ribozymes, antisense cDNAs, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, any Isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. Examples of nucleic acid molecules include mRNA splicing variants. The nucleic acid may be naturally occurring, eg, DNA or RNA, or may be a synthetic analog as is known in the art. Such analogs demonstrate stability under assay conditions and are therefore suitable as probes. Nucleic acid molecules may also include modified nucleic acid molecules, such as methylated nucleic acid molecules and nucleic acid molecule analogs. Purine and pyrimidine analogs are known in the art.

  The nucleic acids of the invention are useful as hybridization probes for the differential identification of tissue (s) or cell type (s) present in a biological sample. Fragments of full-length MGD-CSF variants serve as hybridization probes for cDNA libraries for isolating full-length genes and for isolating other genes with high sequence similarity or similar biological activity. Can be used. This type of probe may have at least 30 bases and may include, for example, 50 or more bases. This probe is also used in screening procedures to identify cDNA clones corresponding to full-length transcripts, and genomic clone (s) containing the complete MGD-CSF gene, including regulatory and promoter regions, exons and introns. Can be used. An example of such a screening involves isolating the coding region of the MGD-CSF gene by using a known nucleic acid sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having sequences complementary to the genes of the present invention can be used to screen human cDNA, genomic DNA or mRNA libraries to identify complementary library components.

The invention further relates to a polynucleotide that hybridizes to a sequence as described herein above, if there is at least 91%, at least 92%, or at least 95% identity between the sequences. The present invention relates to polynucleotides that hybridize under stringent conditions to the polynucleotides described herein above. Stringent conditions generally include conditions in which hybridization occurs only when there is at least 95%, or at least 97% identity between the sequences. For example, 50% formamide, 5 × SSC (150 mM NaCl, 15 mM
Overnight at 42 ° C. in a solution containing 50 mM sodium phosphate (pH 7.6), 5 × Denhart solution, 10% dextran sulfate, and 20 μg / ml denatured sheared salmon sperm DNA. The subsequent incubation of the filter at about 65 ° C. in 0.1 × SSC constitutes stringent conditions.

  Polynucleotides that hybridize to the polynucleotides shown in the Tables and Sequence Listing can encode polypeptides that substantially retain the same biological function or activity as the mature polypeptide. Alternatively, the polynucleotide may have at least 20 bases, at least 30 bases or at least 50 bases that hybridize to and have identity to the polynucleotide of the invention, and this may be It may or may not retain the same biological function or activity. Accordingly, the present invention provides at least 70% identity, at least 80% identity, at least 90% identity, or at least 95% identity to a polynucleotide encoding a polypeptide set forth in the sequence listing. As well as fragments thereof having at least 30 bases or at least 50 bases and to the polypeptides encoded by such polynucleotides.

  Using the information provided herein, eg, the nucleotide sequences shown in the tables and sequence listings, the nucleic acid molecules of the invention encoding MGD-CSF polypeptides can be prepared using standard cloning and screening procedures, such as starting materials. Can be obtained using the procedure for cloning cDNA using mRNA as

(Modified and mutant polynucleotides)
The invention further relates to variants of the nucleic acid molecules of the invention that encode portions, analogs or derivatives of the MGD-CSF molecule. Variants are described in, for example, Gene II, Lewin, Ed. B, John Wiley & Sons, New York (1985). It can exist naturally, such as a natural allelic variant, such as one of the other forms. Non-naturally occurring variants can be generated using mutagenesis techniques known in the art.

  Such variants include variants produced by nucleotide substitutions, deletions or additions. This substitution, deletion or addition may comprise one or more nucleotides. This variant may be altered in the coding region, non-coding region, or both. Changes in the coding region can result in conservative or non-conservative amino acid substitutions, deletions or additions. These may take the form of silent substitutions, additions or deletions that do not alter the properties or activity of the desired MGD-CSF protein, or portion thereof.

  In certain embodiments, the invention provides nucleic acid molecules encoding mature proteins, including, for example, proteins having a truncated signal peptide or leader sequence, as shown in the sequence listing. Another embodiment is an isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence that is at least 70% identical, at least 80% identical, at least 90% identical or at least 95% identical to a polynucleotide from the sequence listing , A polypeptide encoded by a polynucleotide set forth in the Sequence Listing, a polypeptide set forth in the Sequence Listing, or a biologically active fragment of any of these.

  A polynucleotide having a nucleotide sequence that is at least, eg, 95% identical to a reference nucleotide sequence encoding an MGD-CSF polypeptide, except that it may include up to 5 point mutations per 100 nucleotides of this reference nucleotide sequence. A polynucleotide whose nucleotide sequence is identical to a reference sequence. In other words, in order to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to the reference nucleotide sequence, up to 5% of the nucleotides in this reference sequence are either deleted or another nucleotide. It may be substituted or multiple nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These variations of the reference sequence are individually distributed between the nucleotides in the reference sequence, either at the 5 'or 3' end position of the reference nucleotide sequence, or between their terminal positions, or It may occur in one or more consecutive groups within the reference sequence.

  In practice, whether any particular nucleic acid molecule is, for example, at least 70%, 80%, 90% or 95% identical to the nucleotide sequence shown in the sequence listing is determined by the Bestfit program (WisconsinSequence Analysis Package). , Version 8 for Unix (R), Genetics Computer Group, Madison, Wis.) Can be conveniently determined using known computer programs. Bestfit finds the optimal segment of homology between two sequences using the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981). When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the present invention, the parameters will of course be identical. The percentage is calculated over the entire length of the reference nucleotide sequence and is set to allow gaps in homology up to 5% of the total number of nucleotides in the reference sequence.

  This application is at least 70%, 80%, 90% or 95% identical to the nucleic acid sequence shown in the sequence listing, regardless of whether the nucleic acid molecule encodes a polypeptide having MGD-CSF activity. Relates to nucleic acid molecules. Even if a particular nucleic acid molecule does not encode a polypeptide having MGD-CSF activity, one skilled in the art knows how to use the nucleic acid molecule as, for example, a hybridization probe, or primer for polymerase chain reaction (PCR). is there. Uses of the nucleic acid molecules of the invention that do not encode a polypeptide having MGD-CSF activity include, inter alia, isolating the MGD-CSF gene or allelic variant thereof in a cDNA library; and Verna et al., Human Chromosomes. : Hybridization to metaphase chromosomal breaks to predict the exact chromosomal location of the MGD-CSF gene as described in: A Manual of Basic Technologies, Pergamon Press, New York (1988) (eg, fluorescence in situ hybridization ( FISH)); and Northern blot analysis to detect MGD-CSF mRNA expression in specific tissues.

  The application also provides a nucleic acid molecule having a sequence that is at least 70%, 80%, 90% or 95% identical to a nucleic acid sequence in the sequence listing, wherein the polypeptide has MGD-CSF polypeptide activity, i.e. a specific Relates to a nucleic acid molecule that encodes a polypeptide that exhibits an activity that is identical or similar to the activity of the MGD-CSF polypeptide of the invention as measured in a biological assay. For example, the MGD-CSF polypeptides of the invention can stimulate immune cell proliferation, inhibit tumor growth, and / or kill tumor cells.

  Due to the degeneracy of the genetic code, one skilled in the art will recognize that many nucleic acid molecules having a sequence that is at least 70%, 80%, 90% or 95% identical to the nucleic acid sequence of the nucleic acid sequence shown in the sequence listing. And immediately recognize that it encodes a polypeptide having MGD-CSF polypeptide activity. Indeed, since multiple degenerate variants of these nucleotide sequences encode the same polypeptide, this will be apparent to one of ordinary skill in the art without performing the comparative assay described above. It will be appreciated by those skilled in the art that a substantial number of nucleic acid molecules that are not degenerate variants also encode a polypeptide having MGD-CSF polypeptide activity, and as will be described further below, We are well aware of amino acid substitutions that are unlikely to affect or significantly affect protein function (eg, replacing one aliphatic amino acid with a second aliphatic amino acid).

(Vector and host cell)
The invention also relates to vectors containing the isolated nucleic acid molecules of the invention, host cells genetically engineered with recombinant vectors, and production of MGD-CSF polypeptides or fragments thereof by recombinant techniques. This includes a recombinant vector containing, for example, a nucleic acid construct encoding a secretory leader sequence (see, eg, the Sequence Listing; the secretory leader may be a collagen secretion leader), and the selected heterologous poly Host cells genetically engineered with peptides and recombinant vectors are provided. The vector may be, for example, a phage, plasmid or viral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral transmission occurs only in complementing host cells. The vector of the present invention may comprise a Kozak sequence (Lodish et al., Molecular Cell Biology, 4th edition, 1999). The vectors of the present invention may also contain an ATG start codon of the sequence of interest.

  The polynucleotide can be linked to a vector containing a selectable marker for propagation in the host. In general, plasmid vectors are introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transformed into a host cell.

  DNA inserts are suitable promoters, such as the λ phage PL promoter, to name a few; It may be operably linked to the E. coli lac, trp, phoA and tac promoters; the SV40 early and late promoters; and the retroviral LTR promoter. Other suitable promoters are known to those skilled in the art. The expression construct further includes sites for transcription initiation, termination and transcription regions, and a ribosome binding site for translation. The coding portion of the transcript expressed by this construct may include a translation initiation codon with an initiation and termination codon (UAA, UGA or UAG) appropriately located at the end of the polypeptide to be translated.

  The present invention provides expression of a gene of interest in animals, including humans, particularly under the control of a promoter that functions in the liver. A hydrodynamic-based procedure for tail vein injection (Zhang et al., Hum. Gene Ther. 10: 1735 (1999)) has been demonstrated to transfect cells with the gene of interest. The present invention also provides for manipulation of the level of gene expression by controlling the amount and frequency of intravascular DNA administration. The invention further provides a promoter that functions to express a gene in the liver.

One large family of proteins expressed in the liver is the cytochrome P450 protein family. These proteins are a group of heme-thiolate monooxygenases that often undergo various oxidation reactions as part of the body's mechanism to dispose of harmful substances by making them more water soluble. Most of the body's total amount of cytochrome P450 protein is found in the liver, specifically in the microsomes of hepatocytes. There are over a thousand different cytochrome P450 proteins. However, only 49 genes and 15 pseudogenes have been sequenced in humans. In humans, cytochrome P450 3A4 has been identified as the most important cytochrome P450 protein in oxidative metabolism. This is the most common cytochrome P450 protein in the body and is an inducible protein.

  Expression of a gene in the liver and any other site where this promoter is active by operably linking the promoter sequence of a gene expressed in the liver, eg, the promoter sequence of any cytochrome P450 protein to the gene of interest Can be brought about. The present invention includes but is not limited to cytochrome P450 genes, such as cytochrome P450 3A4; c-jun; jun-b; c-fos; c-myc; serum amyloid A; apolipoprotein B editing catalytic subunit liver) regenerative factors; such as LRF-1 signaling factor and transcriptional activator, such as STAT-3; serum alkaline phosphatase (SAP); insulin-like growth factor binding protein such as IGFBP-1; cyclin D1; Active protein-1; CCAAT enhancer core binding protein; β-ornithine decarboxylase; liver regenerating phosphatase-1 f regenerating river-1); early growth response gene-1; hepatocyte growth factor; hemopexin; insulin-like growth factor (IGF) such as IGF2; hepatocyte nucleus family 1; hepatocyte nucleus family 4; liver Includes promoters that function to express genes including cellular Arg-Ser-rich domain containing proteins; glucose 6-phosphatase; and acute phase proteins such as serum amyloid A and serum amyloid P (SAA / SAP).

  As shown in Table 1 and Example 9, MGD-CSF was operably linked to the MGD-CSF with the cytochrome P450 3A4 promoter sequence and the resulting construct injected into the tail vein of mice. And the accompanying increase in monocyte production by mice. Accordingly, the present invention provides a therapeutic molecule of the present invention that is delivered in vivo. The present invention can be used to deliver naked DNA in the presence or absence of a pharmaceutically acceptable carrier, or vector DNA having a sequence of interest. Methods for assessing the function of the molecules of the invention delivered in vivo are known in the art and some are described herein.

  As indicated, the expression vector may include at least one selectable marker. Such markers include dihydrofolate reductase, G418 or neomycin resistance, and E. coli for eukaryotic cell cultures. For culture in E. coli and other bacteria, genes for tetramycin, kanamycin or ampicillin resistance are included. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as E. coli. E. coli, Streptomyces, and Salmonella typhimurium cells; fungal cells such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293 and Bowes melanoma cells; and plant cells It is done. Appropriate culture media and conditions for the above host cells are known in the art.

  A selectable marker is a gene that confers a phenotype on a cell that expresses the marker so that the cell can be identified under appropriate conditions. In general, selectable markers allow the selection of transformed cells based on the ability of the transformed cells to grow in the presence or absence of compounds or other factors that inhibit essential cell function. Thus, appropriate markers confer drug resistance or sensitivity to drugs, and if cells transfected with a molecule encoding a selectable marker are grown in an appropriate selective medium, do they give color to the cells? Alternatively, a gene encoding a protein that imparts a change in the characteristics of the antigen can be mentioned. For example, selectable markers include cytotoxicity markers and drug resistance markers, which are selected by the ability of cells to grow on media containing one or more cytotoxins or drugs; auxotrophic markers A marker by which a cell is selected for the ability of the cell to grow in a defined medium in the presence or absence of specific nutrients or supplements such as thymidine and hypoxanthine; a metabolic marker, eg, the cell is the only carbon Markers selected for their ability to grow in defined media containing the appropriate sugar as the source, as well as markers that confer the ability of the cells to form colored colonies on the chromogenic substrate or to fluoresce the cells It is done.

  Among the vectors suitable for use in bacteria are pQE70, pQE60 and pQE-9 available from Qiagen, Mississauga, Ontario, Canada; pBS vectors available from Stratagene (La Jolla, Calif.), Pagescript vector, Bluescript vector , PNH8A, pNH8A, pNH6a, pNH18A, pNH46A; and prc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia (Peapack, NJ). Particularly suitable eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be apparent to those skilled in the art.

Other suitable vectors include vectors that use the pTT vector backbone (FIGS. 2, 3 and Durocher et al., Nucl. Acids Res. 30 (2002)). In short, the pTT vector backbone can be prepared, for example, by obtaining pIRESpuro / EGFP (pEGFP) and pSEAP basic vector (s) from Clontech (Palo Alto, Calif.) And pcDNA3.1, pcDNA3.1 / Myc- (His) ₆ (6 × His tag disclosed in SEQ ID NO: 277) and pCEP4 vector can be obtained, for example, from Invitrogen. As used herein, a pTT5 backbone vector can generate pTT5-Gateway and can be used to transiently express proteins in mammalian cells. The pTT5 vector may be derivatized, for example, into pTT5-A, pTT5-B, pTT5-D, pTT5-E, pTT5-H and pTT5-I. As used herein, a pTT2 vector can generate a construct for proper expression in a mammalian cell line.

  The expression vector pTT5 allows extrachromosomal replication of cDNA driven by the cytomegalovirus (CMV) promoter. The plasmid vector pCDNA-pDEST40 is a Gateway compatible vector that can utilize the CMV promoter for high level expression. SuperGlo GFP variant (sgGFP) can be obtained from Q-Biogene (Carlsbad, CA). Preparation of the pCEP5 vector can be accomplished by removing the CMV promoter and polyadenylation signal of pCEP4 by sequential digestion and self-ligation with SalI and XbaI enzymes resulting in plasmid pCEP4Δ. The GblII fragment from pAdCMV5 (Massie et al., J. Virol., 72: 2289-2296 (1998)) encodes a CMV5-poly (A) expression cassette linked to a BglII-linearized pCEP4Δ, pCEP5 vector Is obtained.

The pTT vector can be prepared by deleting the hygromycin (BsmI and SalI excision followed by filling and ligation) and EBNA1 (ClaI and NsiI excision followed by filling and ligation) expression cassettes. The ColEI origin (FspI-SalI fragment containing the 3 ′ end of the β-lactamase ORF) may be replaced with an FspI-SalI fragment from pcDNA3.1 containing the pMBI origin (the same 3 ′ end of the β-lactamase ORF). Myc- (His) ₆ (6 × His tag disclosed as SEQ ID NO: 277) C-terminal fusion tag was obtained after in-frame ligation in pcDNA3.1 / Myc-His digested with HindIII and EcoRV followed by SEAP (pSEAP-basic Derived HindIII-HpaI fragment). The plasmid was subsequently expanded into E. coli grown in LB medium. may be grown in E. coli (DH5α) and purified using a MAXIprep column (Giagen, Mississauga, Ontario, Canada). For quantification, the plasmid can subsequently be diluted in 50 mM Tris-HCl pH 7.4 and the absorbance measured at 260 nm and 280 nm. Plasmid preparations with about 1.75 to about 2.00 _A 260 _{/ A 280} ratio between is suitable.

Introduction of the construct into the host cell can be accomplished by calcium phosphate transfection, DEAE dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection or other methods. Such a method is described in Sambrook, J. et al. (2001) Molecular Cloning, A
Laboratory Manual. It is described in many standard laboratory manuals such as the third edition, Cold Spring Harbor Laboratory Press.

  The polypeptide may be expressed in a modified form, such as a fusion protein, and may include additional heterologous functional regions as well as secretion signals. For example, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of this polypeptide to improve stability and persistence in the host cell during purification or subsequent handling and storage. May be. A peptide moiety may also be added to this polypeptide to facilitate purification. Such regions can be removed prior to final preparation of the polypeptide.

(Polypeptide)
The present invention further provides nucleotide sequences as shown in the tables and sequence listings, eg, full length polypeptide, exon 4, mature polypeptide and fragment TRLRAQ (SEQ ID NO: 11), respectively, between exon 3 and exon 4 of MGD-CSF. An isolated MGD-CSF polypeptide comprising the amino acid sequence encoded by SEQ ID NOs: 7, 8, 9 and 11 corresponding to The present invention provides novel uses for novel polypeptides and related polypeptides, as shown in SEQ ID NOs: 10 and 12.

  The present invention provides a secreted protein that can be directed to the ER, secretory vesicles or extracellular space as a result of a secretory leader, signal peptide or leader sequence, and a protein that is released into the extracellular space without the need to include a signal sequence . If the secreted protein is released into the extracellular space, it may undergo extracellular processing to the mature polypeptide. Release into the extracellular space can occur by many mechanisms, including exocytosis and proteolytic cleavage.

  As shown in FIG. 8A, the MGD-CSF polypeptide can be removed from the recombinant cell culture and isolated by well-known methods. Such methods include ammonium sulfate and ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. Can be mentioned. High performance liquid chromatography (HPLC) may be used for purification. Polypeptides of the present invention, whether isolated directly or cultured, are purified from natural sources including body fluids, tissues and cells; products of chemical synthesis procedures; and, for example, bacteria, yeast, Includes products produced by recombinant techniques from prokaryotic or eukaryotic hosts, including higher plant, insect and mammalian cells. Depending on the host used in the recombinant production procedure, the polypeptides of the invention may or may not be glycosylated. Furthermore, the polypeptides of the present invention may also contain an initial modified methionine residue, in some cases as a result of a host-mediated process. Thus, it is well known in the art that the N-terminal methionine encoded by the translation initiation codon is generally efficiently removed from any protein after translation in eukaryotic cells. The N-terminal methionine on most proteins is also efficiently removed in most prokaryotes, but for some proteins this prokaryotic removal process is the nature of the amino acid to which the N-terminal methionine is covalently attached. Dependent on being inadequate.

  Typically, the heterologous polypeptide may or may not be modified, expressed as described above, or as a fusion protein, and includes not only a secretion signal but also a secretion leader sequence. May be included. The secretory leader sequence of the present invention is directed to a specific protein relative to the endoplasmic reticulum (ER). This ER separates membrane-bound proteins from other proteins. Once localized to the ER, the protein can be further directed to the Golgi apparatus for distribution to vesicles, including secretory vesicles; plasma membranes; lysosomes; and other organelles.

  Proteins targeted to the ER by secretory leader sequences can be released into the extracellular space as secreted proteins. For example, vesicles containing secreted proteins may be fused with cell membranes and their contents can be released into the extracellular space via exocytosis. Exocytosis can occur constitutively or upon receipt of an attraction signal. In the latter case, the protein can be stored in secretory vesicles (or secretory granules) until exocytosis is triggered. Similarly, proteins present on the cell membrane can also be secreted into the extracellular space by proteolytic cleavage of the linker that holds the protein to the membrane.

  In addition, peptide moieties and / or purification tags can be added to the polypeptide to facilitate purification. Such regions can be removed prior to final preparation of the polypeptide. Addition of a peptide moiety to a polypeptide to cause secretion or excretion, improve stability, and facilitate purification, among other reasons, is well known and routine in the art. Technology. Suitable purification tags include, for example, V5, HISX6 (SEQ ID NO: 277), HISX8 (SEQ ID NO: 278), avidin and biotin.

  The present invention provides fusion proteins comprising heterologous regions derived from immunoglobulin that are useful for stabilizing and purifying proteins. Among other things, the addition of a peptide moiety to a polypeptide to produce secretion or excretion, to improve stability, and to facilitate purification is a technique well known and routine in the art. is there. For example, EP 0 464 533 (Canadian counterpart 2045869) discloses a fusion protein comprising various portions of the constant region of an immunoglobulin molecule together with another human protein or part thereof. In many cases, the Fc portion of the fusion protein is advantageous for use in therapy and diagnosis, thus, for example, resulting in improved pharmacokinetic properties (European Patent 0 232 262). On the other hand, for some applications, it is desirable that the fusion protein be capable of deleting the Fc portion after being expressed, detected and purified in the advantageous manner described. This is especially true when the Fc protein proves to interfere with therapeutic and / or diagnostic uses, for example when the fusion protein is to be used as an antigen for immunization. For example, in drug development, human proteins, such as hIL-5, are fused with Fc moieties for the purpose of high-throughput screening assays to identify antagonists of hIL-5. Bennett et al. Molec. Recog. 8: 52-58 (1995) and Johanson et al., J. MoI. Biol. Chem. 270: 9459-9471 (1995).

  The polypeptides of the invention may be provided in isolated form and may be substantially purified as described above. A recombinantly produced version of the MGD-CSF polypeptide described herein is also substantially isolated according to the one-step method described in, for example, Smith and Johnson, Gene, 67: 31-40 (1988). Also good. The polypeptides of the present invention may further be isolated from natural or recombinant sources using the anti-MGD-CSF antibodies of the present invention produced using methods well known in the art.

  The polypeptides described herein can be used in the presence of ions or factors that assist in the folding of the molecule or assist in the dimerization or trimerization of the molecule, as is customary in the art. It can be purified or isolated. For example, cofactors can be added to promote physiological folding or polymerization.

  Additional polypeptides of the present invention include polypeptides having at least 70%, 80%, 90% or 95% identity to the above polypeptides. Polypeptides of the present invention also include polypeptides that are at least 70%, 80%, 90% or 95% identical to the polypeptides encoded by the nucleic acid sequences in the sequence listing.

  The percent identity of two polypeptides can be measured by a similarity score determined by comparing the amino acid sequences of the two polypeptides using the Bestfit program using the default settings for similarity determination. Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematicas 2: 482-489 (1981) to find the optimal segment of similarity between two sequences.

  For example, a polypeptide having an amino acid sequence that is at least 95% identical to a reference amino acid sequence of an MGD-CSF polypeptide means that the amino acid sequence of the polypeptide has a maximum per 100 amino acids of each of the reference polypeptides. A polypeptide whose polypeptide amino acid sequence is identical to this reference sequence except that it may contain up to five amino acid modifications. In other words, to obtain a polypeptide having an amino acid sequence that is at least 95% identical to the reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence are deleted or another An amino acid may be substituted, or multiple amino acids, up to 5% of the total amino acid residues in the reference sequence may be inserted into this reference sequence. These changes in the reference sequence can occur either at the amino-terminal or carboxy-terminal position of the reference amino acid sequence or between the terminal positions, individually between residues of this reference sequence, or It may be distributed in one or more consecutive groups within this reference sequence.

  In practice, any particular polypeptide may be, for example, at least 70%, 80%, 90% or 95% to the amino acid sequence encoded by the nucleic acid sequence shown in the sequence listing or to the polypeptide sequence. Whether they are the same or not can be conveniently determined using a known computer program such as the Bestfit program. When using Bestfit or other sequence alignment programs to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the present invention, the parameter will of course be a percentage identity. Calculated over the entire length of this reference amino acid sequence and is set to allow gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence.

(Modified and mutant polypeptides)
Protein manipulation can be used to improve or alter the characteristics of the MGD-CSF polypeptides of the invention. Recombinant DNA technology known to those skilled in the art can be used to generate new mutant proteins or “mutant proteins (muteins)” including single or multiple amino acid substitutions, deletions, additions or fusion proteins. Such modified polypeptides can exhibit desirable properties, such as enhanced activity or increased stability. Furthermore, they can be purified at higher yields than the corresponding native polypeptide, at least under certain purification and storage conditions, and exhibit good solubility.

(N-terminal and C-terminal deletion mutants)
For example, for many proteins, including the extracellular domain of membrane-bound proteins or mature secreted proteins, one or more amino acids can be removed from the N-terminus or C-terminus without substantial loss of biological function in the art. It is known that it can be deleted. For example, Ron et al. Biol. Chem. 268: 2984-2988 (1993) reported a modified KGF protein having heparin binding activity even if 3, 8 or 27 amino terminal amino acid residues were deleted.

  However, even when deletion of one or more amino acids from the N-terminus of the protein results in alteration or loss of one or more biological functions of the protein, other biological activities are still retained. obtain. Accordingly, the ability of a truncated protein to induce and / or bind to an antibody that recognizes the complete or mature form of the protein is generally less than a majority of the residues of the complete or mature protein. Retained when removed from the end. Whether a particular polypeptide lacking the N-terminal residue of the complete protein retains such immunological activity can be determined by routine methods described herein, and so on. Otherwise it is known in the art. Accordingly, the present invention further provides a polypeptide in which one or more residues are deleted from the amino terminus of the amino acid sequence of the MGD-CSF molecule as shown in the sequence listing.

  Similarly, many examples of biologically functional C-terminal deletion muteins (muteins) are known. For example, interferon γ increases the activity by about 10 times when 8 to 10 amino acid residues are deleted from the carboxy terminus of this protein. For example, Dobeli et al. See Biotechnology 7: 199-216 (1988).

  However, even if deletion of one or more amino acids from the C-terminus of the protein results in alteration of loss of one or more biological functions of the protein, other biological activities can still be retained. Thus, the ability of a truncated protein to induce and / or bind to an antibody that recognizes the complete or mature form of the protein is generally less than a majority of the residues of the complete or mature protein. Retained when removed from the end. Whether a particular polypeptide lacking the C-terminal residue of the complete protein retains such immunological activity can be determined by routine methods described herein, and so on. Otherwise it is known in the art.

(Mutein from cysteine to serine (mutein))
The MGD-CSF sequence contains seven cysteine residues located at amino acid positions 35, 167, 176, 178, 179, 190 and 198. In certain embodiments, the invention provides a mutant MGC34647 molecule having a serine mutated to cysteine. These variants are shown in Table 1 and the Sequence Listing and are named SEQ ID NOs: 258-271. The collagen signal peptide set forth in SEQ ID NO: 14 was used to improve secretion of the expressed polypeptide. As shown in Table 1 and FIG. 8B and illustrated in more detail in Example 18, the cysteines at positions 35, 167, 176, 178, 179, 190 and 198 were each replaced with serine. These constructs may be cloned into any suitable vector as known in the art, for example, the pTT5-G vector.

  By analyzing these muteins (muteins), the disulfide bond pattern of MGD-CSF is understood and improved properties such as improved expression and secretion from mammalian cells, purified protein Reduced aggregation, and E.I. When expressed in E. coli, proteins having the ability to produce active recombinant MGD-CSF can be identified.

(Other mutants)
It will also be appreciated by those skilled in the art that in addition to the terminally deleted forms of the proteins discussed above, some amino acid sequences of MGD-CSF polypeptides can be altered without significant effects on the structure or function of the protein. . If such differences in sequence are intended, it should be remembered that there are important regions on the protein that determine activity.

  Accordingly, the present invention further encompasses variants of MGD-CSF polypeptides that exhibit substantial MGD-CSF polypeptide activity or that include regions of MGD-CSF proteins, such as the protein portions discussed below. . Such mutants include deletions, insertions, inversions, repeats and type substitutions selected according to general rules known in the art that have little effect on activity. For example, guidance on how to create phenotypically silent amino acid substitutions is provided in Bowie et al., Science 247: 1306-1310 (1990), which authors have two main approaches to studying amino acid sequence tolerance to changes. That there is a good approach. This first method relies on an evolutionary process, where mutations are allowed or rejected by natural selection. The second approach uses genetic engineering to induce amino acid changes at specific positions in the cloned gene and selection, or to screen and identify sequences that maintain function.

  These studies surprisingly report that the protein is resistant to amino acid substitutions. The authors further indicate which amino acid changes are likely to be tolerated at specific amino acid positions in the protein. For example, most buried amino acid residues require nonpolar side chains, but the characteristics of surface side chains are generally rarely conserved. Other such phenotypically silent substitutions are described in Bowie et al., Supra and references cited therein. Typically shown as conservative substitutions are substitutions of one amino acid with another, especially the aliphatic amino acids Ala, Val, Leu and Ile; exchange of hydroxyl residues Ser and Thr, acidic residues Asp And Glu exchange, substitution between amide residues Asn and Gln, exchange of basic residues Lys and Arg, and substitution between aromatic residues Phe and Tyr.

  Thus, a polypeptide, sequence fragment or derivative or analog encoded by a sequence listing polypeptide or a sequence listing nucleic acid sequence is (i) one or more amino acid residues replaced with conserved or non-conserved amino acid residues Such substituted amino acid residues may or may not be amino acid residues encoded by the genetic code; (ii) one or more amino acid residues Having a substituent; (iii) the mature polypeptide is fused with another compound, eg, a compound that increases the half-life of the polypeptide (eg, polyethylene glycol); or (iv) an additional amino acid is Polypeptides of the above forms, such as IgG Fc fusion region peptides, leader sequences or secretory sequences, forms of the above Sequences are used to purify the polypeptide, or may be those to be fused with the precursor protein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

  Thus, the MGD-CSF polypeptides of the present invention may comprise one or more amino acid substitutions, deletions or additions derived from either natural variants or human manipulation. As shown, these changes can be minor changes, such as conservative amino acid substitutions that do not significantly affect protein folding or activity. Conservative amino acid substitutions include: aromatic substitutions Phe, Trp and Tyr; hydrophobic substitutions Leu, Iso and Val; polar substitutions Glu and Asp; basic substitutions ARg, Lys and His; acidic substitutions Asp and Glu And the small amino acid substitutions Ala, Ser, Thr, Met and Gly.

  Amino acids essential for the function of the MGD-SCF polypeptide can be identified by methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis. See, for example, Cunningham and Wells, Science, 244: 1081-1085 (1989). The latter procedure introduces a single alanine mutation. The resulting mutant molecules are then tested for biological activity such as receptor binding or for in vitro or in vivo proliferative activity.

  Of particular interest is the substitution of charged amino acids with other charged or neutral amino acids that can produce proteins with highly desirable improved characteristics, such as reduced aggregation. Aggregation can not only reduce activity, but can also be a problem when preparing pharmaceutical formulations. Because, for example, aggregates can be immunogenic, Pincard et al., Clin. Exp. Immunol. 2: 331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10: 307-377 (1993)).

  Amino acid substitutions can alter the selectivity of ligand binding to cell surface receptors. For example, Ostade et al., Nature, 361: 266-268 (1993) describe mutations that result in selective binding of TNF-α to only one of the two known types of TNF receptors. Sites that are important for ligand-receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling, see, for example, Smith et al. Mol. Biol. , 224: 899-904 (1992) and de Vos et al., Science, 255: 306-312 (1992).

(Epitope holding part)
As described in detail below, the polypeptides of the invention are used to enhance MGD-SCF protein function in assays to detect MGD-SCF protein expression, as also described below. Alternatively, polyclonal and monoclonal antibodies that are useful as agonists and / or antagonists that can be inhibited can be raised. These polypeptides can also be used in a yeast two-hybrid system for capturing MGD-SCF protein binding, which are also candidate agonists and antagonists according to the present invention. The yeast two-hybrid system is described in Fields and Song, Nature, 340: 245-246 (1989).

  In another aspect, the present invention provides a polypeptide comprising one or more epitope-bearing portions of the polypeptide of the present invention. The present invention provides polyclonal antibodies specific for MGD-CSF and provides MGD-SCF with at least two antigenic epitopes. The epitope of this polypeptide part is an immunogenic or antigenic epitope of the polypeptide of the present invention. An immunogenic epitope is a portion of a protein that elicits an antibody response when the entire protein is provided as an immunogen. On the other hand, the region of the protein molecule to which the antibody can bind is an antigenic epitope. The number of immunogenic epitopes in a protein is generally less than the number of antigenic epitopes. See, for example, Geysen et al., Proc. Natl. Acad. Sci. 81: 3998-4002 (1983).

  With respect to the selection of a polypeptide carrying an antigenic epitope (ie, a polypeptide comprising a region of a protein molecule to which an antibody can bind), a relatively short synthetic peptide that mimics part of the protein sequence is partly mimicked. It is well known to those skilled in the art that antisera that interact with the engineered protein can be routinely raised. See, for example, Sutcliffe et al., Science, 219: 660-666 (1983). Peptides capable of eliciting protein-reactive sera are frequently presented in the primary sequence of proteins, can be characterized by a simple set of chemical rules, and are immunodominant regions of intact proteins (ie, immunogenic Epitope) or amino or carboxyl terminus. Accordingly, the antigenic epitope-bearing peptides and polypeptides of the present invention are useful for raising antibodies, including monoclonal antibodies that specifically bind to the polypeptides of the present invention. See, for example, Wilson et al., Cell, 37: 767-778 (1984). The epitope-bearing peptides and polypeptides of the present invention can be generated by any conventional method. For example, Houghten, Proc. Natl. Acad. Sci. 82: 5131-5135 (1985) and U.S. Pat. No. 4,631,211 (1986).

  The epitope-bearing peptides and polypeptides of the present invention can be used to induce antibodies according to methods well known in the art. For example, Bittle et al. Gen. Virol. 66: 2347-2354 (1985). The immunogenic epitope-bearing peptides of the present invention that are part of a protein that elicits an antibody response are identified according to methods known in the art when the entire protein is an immunogen. For example, a general method for detecting or determining the sequence of a monomer (amino acid or other compound) that is a topological equivalent of an epitope (mimotope) that is complementary to a specific antigen binding site (paratope) of an antibody of interest U.S. Pat. No. 5,194,392 (1990), which describes U.S. Pat. More generally, U.S. Pat. No. 4,433,092 (1989) detects a sequence of monomers that are topological equivalents of a ligand that is complementary to the ligand binding site of a particular receptor of interest. Describes how to determine. Similarly, US Pat. No. 5,480,971 (1996) describes linear C1-C7-alkyl peralkylated oligopeptides, and sets and libraries of such peptides and, for example, acceptor molecules of interest. Disclosed are methods for using such oligopeptide sets and libraries to determine the sequence of peralkylated oligopeptides that bind to. Thus, non-peptide analogs of the epitope-bearing peptides of the invention can also be routinely made by these methods.

(Fusion molecule)
As will be appreciated by those skilled in the art, the MGD-CSF polypeptide of the present invention, and the above epitope-bearing fragments thereof, can be combined with a heterologous polypeptide to produce a chimeric polypeptide. These fusion proteins facilitate purification and show increased half-life in vivo. This has been reported, for example, in chimeric proteins consisting of the first two domains of a human CD4 polypeptide and various domains of the constant region of a mammalian immunoglobulin heavy chain or light chain, eg, European Patent 0 394 827 Traunecker et al., Nature, 331: 84-86 (1988). Fusion proteins having a disulfide bond dimer structure resulting from an IgG moiety are also described, for example, in Fountoulakis et al. Biochem. 270: 3958-3964 (1995), may be more efficient in binding and neutralizing other molecules than monomeric MGD-CSF protein or protein fragment alone. Suitable chemical moieties for derivatization of heterologous polypeptides are described herein and are described in US Pat. Nos. 6,686,179 and US Patent Applications 60 / 589,788 and 60 / 654,229. For example, polymers, eg water-soluble polymers, immunoglobulin constant domains, all or part of human serum albumin; fetuin A; fetuin B; leucine zipper domain; tetranectin trimerization domain Mannose binding protein (also known as mannose binding lectin), for example, mannose binding protein 1; and Fc region. Methods for making fusion proteins are well known to those skilled in the art.

  For example, due to the short plasma half-life of unmodified interferon alpha, frequent administration over a long period of time is required to treat viruses and proliferative disorders. Interferon alpha fused to HSA has a longer half-life and requires less administration frequency than unmodified interferon alpha; this half-life is 18 times longer and the clearance rate is about 140 times slower ( Osborn et al., J. Pharmacol. Exp. Ther. 303: 540-548, 2002). Interferon beta fused with HSA also has beneficial pharmacokinetic properties; its half-life has been reported to be 36-40 hours compared to 8 hours with unmodified interferon beta. (Sung et al., J. Interferon Cytokine Res. 23: 25-36, 2003). HSA-interleukin-2 fusion proteins have been reported to have both a long half-life and beneficial biodistribution compared to unmodified interleukin-2. This fusion protein has been observed to target tissues where lymphocytes are present to a greater extent than unmodified interleukin 2, suggesting that the fusion protein exerts greater efficacy. (Yao et al., Cancer Immunol. Immunother. 53: 404-410, 2004).

  Human immunoglobulin G subclass 1 Fc receptors have also been used as fusion proteins for therapeutic molecules. It is recombinantly bound to two soluble p74 tumor necrosis factor (TNF) receptor molecules. This fusion protein has been reported to have a longer circulating half-life than the monomeric soluble receptor and to inhibit TNFα-induced pro-inflammatory activity in the joints of patients with rheumatoid arthritis (Goldenberg, Clin. Ther., 21: 75-87, 1999). This fusion protein has been used clinically to treat rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis and ankylosing spondylitis (Nanda and Bathon, Expert Opin. Pharmacother. 5: 1175-1186, 2004). ).

  Polymers, such as water-soluble polymers, are useful as polypeptides in the present invention, and each polymer is attached to the polypeptide, eg, water-soluble as typically found in physiological environments. In the environment of no precipitation. The polymers used in the present invention are pharmaceutically acceptable for the preparation of therapeutic products or compositions. One skilled in the art can select the desired polymer based on such considerations as the polymer / protein conjugate being used therapeutically, and if so, the desired dosage, circulation time and resistance to proteolysis. .

  Suitable clinically acceptable water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), polyethylene glycol propionaldehyde, ethylene glycol / propylene glycol copolymers, monomethoxy-polyethylene glycol, carboxymethyl cellulose, dextran. , Polyvinyl alcohol (PVA), polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, poly (β-amino acid) (either homopolymer or random copolymer ), Poly (n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymer (PPG) and other polyalkylene oxides, polypropylene oxide / ethylene Xoxide copolymers, polyoxyethylated polyols (POG) (eg glycerol) and other polyoxyethylated polyols, polyoxyethylated sorbitol or polyoxyethylated glucose, colonic acids or other carbohydrate polymers, Ficoll Or dextran and mixtures thereof.

  As used herein, polyethylene glycol (PEG) is any form used to derive other proteins such as mono- (C1-C10) alkoxy- or aryloxy-polyethylene glycol. It means to do. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.

  In particular, the modified heterologous polypeptide of the invention can be prepared by attaching a polyamino acid or a branch point amino acid to the polypeptide. For example, a polyamino acid can be a carrier protein that functions to increase the circulating half-life of a polypeptide (in addition to the benefits achieved through a fusion molecule). For the therapeutic purposes of the present invention, such polyamino acids should ideally have no or no neutralizing antigenic response and no other adverse response. Such polyamino acids include serum albumin (eg, human serum albumin), additional antibodies or portions thereof, such as Fc region, fetuin A, fetuin B, leucine zipper nuclear factor erythrocyte inducer-2 (NFE2), neuroretinal leucine. It may be selected from zippers, tetranectin, or other polyamino acids such as lysine. As described herein, the position of attachment of the polyamino acid may be N-terminal or C-terminal, or other position in between, and also a chemical linker moiety for the selected molecule. May be connected by.

  The polymers used herein, such as water soluble polymers, can be any molecular weight polymer and may or may not be branched. Each of the polymers typically has an average molecular weight of about 2 kDa to about 100 kDa (the term “about” means that in the preparation of the polymer some molecules are greater than the stated molecular weight, Show some less). The average molecular weight of each polymer may be from about 5 kDa to about 50 kDa, or from about 12 kDa to about 25 kDa. In general, the higher the molecular weight or the more branched, the higher the polymer: protein ratio. Desired therapeutic profile; for example, sustained release period; effects on biological activity, if any; ease of handling; degree or lack of antigenicity; and other polymers for modified molecules of the invention Depending on the known effects; other sizes may also be used.

  The polymer used in the present invention is typically conjugated to a heterologous polypeptide in view of its effect on the functional domain or antigenic domain of the polypeptide. In general, chemical derivatives can be performed under any suitable conditions used to react proteins with activated polymer molecules. Activating groups that can be used to link the polymer to the active moiety include sulfone, maleimide, sulfhydryl, thiol, triflate, tresylate, azidiline, oxirane and 5-pyridyl.

  The polymers of the present invention are typically attached to a heterologous polypeptide with an alpha (α) or epsilon (ε) amino group of an amino acid, or a reactive thiol group, but the polymer group is subjected to suitable reaction conditions. It is also contemplated that it may be attached to any reactive group of the protein that is sufficiently reactive to attach to the polymer group. Thus, the polymer may be covalently attached to the heterologous polypeptide via a reactive group such as a free amino or carboxyl group. The amino acid residue having a free amino group may include a leucine residue and an N-terminal amino acid residue. Examples of the residue having a free carboxyl group include an aspartic acid residue, a glutamic acid residue, and a C-terminal amino acid residue. Examples of the residue having a reactive thiol include a cysteine residue.

  Each method for preparing a fusion molecule conjugated with a polymer, such as a water-soluble polymer, generally involves (a) combining a heterologous polypeptide and a polymer with the one or more polymers. And (b) a step of obtaining the reaction product. The reaction conditions for each binding may be selected from any condition known in the art or subsequently developed conditions, but the temperature, solvent and pH levels that inactivate the protein to be modified. Must be selected to avoid or limit exposure to reaction conditions such as In general, the optimal reaction conditions for the reaction will optionally be determined based on known parameters and the desired result. For example, the greater the polymer: polypeptide conjugate ratio, the greater the percentage of product conjugated. The optimal ratio (in terms of the effectiveness of the reaction, in that there is no excess unreacted polypeptide or polymer) was selected for the desired degree of derivatization (eg mono-, di-tri-, etc.) It can be determined by factors such as the molecular weight of the polymer, whether the polymer is branched or unbranched, and the reaction conditions used. The ratio of polymer to polypeptide (eg, PEG) generally ranges from 1: 1 to 100: 1. One or more purified conjugates may be prepared from each mixture by standard purification techniques, including dialysis, salting out, ultrafiltration, ion exchange chromatography, gel filtration chromatography and electrophoresis, among others. Good.

  An N-terminal chemically modified protein may be particularly desirable. The polymer is selected by molecular weight, branching, etc., the ratio of polymer to protein (polypeptide or peptide) molecule in the reaction mixture, the type of reaction to be performed, and the method of obtaining the selected N-terminal chemically modified protein Can be done. A method for obtaining an N-terminal chemically modified protein preparation (separating this part from other monoderivatized parts as necessary) from a population of chemically modified protein molecules It may be by purification of a protein substance chemically modified at the N-terminus.

  Selective N-terminal chemical modification can be achieved by reductive alkylation that elicits different reactivity of different types of primary amino groups (lysine vs. N-terminus) available for derivatization in specific proteins. Under appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group-containing polymer is achieved. For example, by reacting the pKa difference between the ε-amino group of the lysine residue and the α-amino group of the N-terminal residue of this protein at a pH that can take advantage of the polymer against the N-terminus of the protein. You may combine selectively. Such selective derivatization controls the binding of the polymer to the protein: conjugation with the polymer occurs preferentially at the N-terminus of the protein and other reactivities such as lysine side chain amino groups. There is no significant modification of the group. Using reductive alkylation, the polymer may be of the type described above and should have a single reactive aldehyde for coupling to proteins. Polyethylene glycol propionaldehyde containing a single reactive aldehyde can also be used.

  In one embodiment, the present invention contemplates that the chemically derivatized polypeptide comprises a mono- or poly (eg 2-4) PEG moiety. Pegylation may be performed by any pegylation reaction known in the art. Methods for preparing PEGylated protein products generally include conditions that allow the protein and polyethylene glycol (eg, a reactive ester or aldehyde derivative of PEG) to be attached to one or more PEG groups. And (b) obtaining the reaction product. In general, the optimal reaction conditions for this reaction are optionally determined based on known parameters and the desired result.

  There are a number of PEG attachment methods available to those skilled in the art. See, for example, European Patent 0 401 384; Malik et al., Exp. Hematol. 20: 1028-1035 (1992); Francis, Focus on Growth Factors, 3 (2): 4-10 (1992); European Patent 0 154 316; European Patent 0 401 384; WO 92/16221; WO 95/34326; and See other publications cited herein regarding pegylation.

  The pegylation step as described herein may be carried out via acylation or alkylation with reactive polyethylene glycol molecules. Thus, protein products according to the present invention include PEGylated proteins in which the PEG group (s) are attached via an acyl group or an alkyl group. Such products may be mono-pegylated or poly-pegylated (eg, those containing 2-6 or 2-5 PEG groups). This PEG group is generally attached to the protein at the alpha or ε amino group of the amino acid, but the PEG group is suitable for proteins that are sufficiently reactive to be attached to the PEG group under appropriate reaction conditions. It is also envisioned that it can be attached to any amino group attached in the same way.

  Pegylation by acylation generally involves reacting an active ester derivative of polyethylene glycol (PEG) with a polypeptide of the invention. For the acylation reaction, the selected polymer (s) typically have a single reactive ester group. Any known or subsequently developed reactive PEG molecule may be used to perform the pegylation reaction. An example of a suitable activated PEG ester is PEG esterified to N-hydroxysuccinimide (NHS). As used herein, acylation is meant to include, but is not limited to, the following types of linkages between therapeutic proteins and polymers: PEG: amide, carbamate, urethane, etc., eg, Chawow, Bioconjugate Chem. 5: 133-140 (1994). The reaction conditions may be selected from any conditions known in the pegylated product or subsequently developed conditions, but avoid conditions such as temperature, solvent and pH that inactivate the polypeptide to be modified. Must.

  PEGylation by acylation generally yields poly-pegylated proteins. The connecting bond may be an amide. The resulting product can be substantially only mono-, 2- or 3-pegylated (eg> 95%). However, some species with a high degree of pegylation can be formed in amounts depending on the specific reaction conditions used. If desired, further purified PEGylated species can be mixed (especially unreacted) by standard purification techniques, including dialysis, salting out, ultrafiltration, ion exchange chromatography, gel filtration chromatography and electrophoresis, among others. Seeds).

  Pegylation by alkylation generally involves reacting a polypeptide with a terminal aldehyde derivative of PEG in the presence of a reducing agent. For reductive alkylation reactions, the selected polymer (s) should have a single reactive aldehyde group. An exemplary reactive PEG aldehyde is polyethylene glycol propionaldehyde, which is water soluble, or a mono-C1-C10 alkoxy- or aryloxy derivative thereof, see, eg, US Pat. No. 5,252,714.

  Furthermore, the heterologous polypeptides of the invention and the epitope-bearing fragments described herein can be combined with a portion of an immunoglobulin (IgG) constant domain to produce a chimeric polypeptide. These particular fusion molecules facilitate purification and show increased half-life in vivo. This has been shown, for example, in chimeric proteins consisting of the first two domains of a human CD4 polypeptide and the various domains of the mammalian immunoglobulin heavy or light chain constant region. For example, European Patent 0 394 827; Traunecker et al., Nature, 331: 84-86 (1988). Fusion molecules with a disulfide-linked dimer structure resulting from the IgG moiety are also more effective in binding and neutralizing other molecules than, for example, monomeric polypeptides or polypeptide fragments alone. For example; Foutoulakis et al., J. MoI. Biocghem. 270: 3958-3964 (1995).

  In another described embodiment, human serum albumin fusion molecules can also be prepared as described herein and as further described in US Pat. No. 6,686,179.

  Furthermore, the polypeptides of the present invention may be fused to a marker sequence such as a peptide that facilitates purification of the fusion polypeptide. The marker amino acid sequence may be, among other things, a hexa-histidine peptide, such as a tag provided in the pQE vector (Qiagen, Mississauga, Ontario, Canada), many of which are commercially available. Gentz et al., Proc. Natl. Acad. Sci. 86: 821-824 (1989), for example, hexa-histidine provides convenient purification of the fusion protein. Another peptide tag useful for purification, the hemagglutination HA tag, corresponds to an epitope derived from influenza hemagglutination protein (Wilson et al., Cell 37: 767 (1984)). Any of these above fusions can be engineered using the polynucleotides or polypeptides of the invention.

(Secretory leader sequence)
In this specification and as demonstrated in US Pat. No. 60 / 647,013, in order for some secreted proteins to be expressed and secreted in large quantities, a secretory leader sequence from another different secreted protein is desired. Is done. The use of a heterologous secretory leader sequence is advantageous in that the resulting mature amino acid sequence of the secreted polypeptide is not altered if the secretory leader sequence is removed in the ER during the secretion process. Furthermore, the addition of a heterologous secretory leader is necessary to express and secrete some proteins.

  Thus, to identify potential strong secretory leader sequence (s) that can be universally used to secrete proteins and express MGD-CSF, Applicants have identified a number of different secretions. Proteins have been cloned and expressed and their expression and secretion levels are measured in the supernatant of cells of human embryonic kidney cell line 293 that has been transformed with adenovirus 5 (Graham et al., J. Gen. Virol. 36:59 (1977)). Several high expression factors and high levels of secreted protein were observed.

  In one embodiment, secretory leader sequences belonging to the secreted protein collagen type IX αI chain length were selected and further tested for their ability to promote expression and secretion when used as heterologous secretory leader sequences. As described herein, the amino acid sequence of the collagen IX αI chain length form, which is a secreted protein, is predicted to be MKTCWKIPVFFFVCFLEPWASA (SEQ ID NO: 14). As described further herein, vectors containing this particular secretion leader are constructed and some proteins remove the secretion leader from the full-length coding sequence and clone them into a vector containing SEQ ID NO: 14. To obtain a secreted protein with a heterologous secretory leader sequence. High expression and secretion of some other selected proteins was also observed.

  The identified secretory leader sequences described herein include, for example, interleukin-9 precursor, T cell growth factor P40, P40 cytokine, triacylglycerol lipase, pancreatic precursor, somatoriverin precursor, vasopressin -Neurophysin 2-copeptin precursor, β-enoendorphin-dinorphine precursor, complement C2 precursor, small inducible cytokine A14 precursor, elastase 2A precursor, plasma serine protease inhibitor precursor, granulocyte macrophage colony stimulating factor Precursor, interleukin 2 precursor, interleukin 3 precursor, α-fetoprotein precursor, α-2-HS-glycoprotein precursor, serum albumin precursor, inter-α-trypsin inhibitor light chain, serum amyloid P component precursor, Polypoprotein A-II precursor, apolipoprotein D precursor, colipase precursor, carboxypeptidase A1 precursor, α-s1 casein precursor, β casein precursor, cystatin SA precursor, follitropin β chain precursor, glucagon precursor Body, complement factor H precursor, histidine-rich glycoprotein precursor, interleukin-5 precursor, α-lactalbumin precursor, Von Ebner gland protein precursor, matrix Gla-protein precursor, α-1- Acid glycoprotein 2 precursor, phospholipase A2 precursor, dendritic cell chemokine 1, statherin precursor, transthyretin precursor, apolipoprotein A-1 precursor, apolipoprotein C-III precursor, apolipoprotein E Precursor, complement component C8 γ chain precursor , Serotransferrin precursor, β-2-microglobulin precursor, neutrophil defensin 1 precursor, triacylglycerol lipase gastric precursor, haptoglobin precursor, neutrophil defensin 3 precursor, tumorigenicity 1 precursor neuroblastoma suppressor, small inducible cytokine A13 precursor, CD5 antigen-like precursor, phospholipid transfer protein precursor, dickkopf related protein-4-precursor, elastase 2B precursor, α-1-acid Glycoprotein 1 precursor, β-2-glycoprotein 1 precursor, neutrophil gelatinase-related lipocalin precursor, C-reactive protein precursor, interferon γ precursor, κ casein precursor, plasma retinol-binding protein precursor, inter Leukin-13 precursor and front It includes any secreted protein shown in the sequence listing.

  The secretory leader sequences, vectors and methods described herein are useful for the expression of various polypeptides, including, for example, receptors such as secreted polypeptides, extracellular proteins, transmembrane proteins and soluble receptors. Examples of such polypeptides include, but are not limited to, cytokines and growth factors such as interleukin 1-18, interferon, lymphokine, hormone, Regulated on Activation, Normal T Expressed and Secreted (RANTES), lymphotoxin-β , Fas ligand, flt-3 ligand, ligand for receptor activator of NF-kappa B (RANKL), soluble receptor, TNF-related apoptosis-inducing ligand (TRAIL), CD40 ligand, Ox40 Ligand, 4-1BB ligand (and other members of the TNF family), derived from thymic stroma Lymphopoietin, stimulating factors such as granulocyte colony stimulating factor (G-CSF) and granulocyte macrophage colony stimulating factor (GM-CSF), inhibitors, mast cell growth factor, hepatocyte growth factor, epidermal growth factor, growth hormone, Tumor necrosis factor (TNF), leukemia inhibitory factor (LIF), oncostatin-M, hematopoietic factors such as erythropoietin and thrombopoietin, and splicing variants of any of these.

Descriptions of some proteins that can be expressed by the present invention include, for example, Human Cytokines: Handbook for Basic and Clinical
Research, Vol. II (Agarwal and Gutterman, edited by Blackwell Sciences, Cambridge Mass, 1998); Ed. W. Thompson; Academic Press, San Diego Calif. 1991).

  Receptors for any of the above proteins may also include, for example, both forms of tumor necrosis factor receptor (referred to as p55 and p75), interleukin-1 receptor (types 1 and 2), interleukin-4 receptor, interleukin-15 Receptor, interleukin-17 receptor, interleukin-18 receptor, granulocyte-macrophage colony stimulating factor receptor, granulocyte colony stimulating factor receptor, receptor for oncostatin-M and leukemia inhibitory factor, receptor activator of NF-κB (receptor activator) of NF-kappaB) (RANK), a receptor for TRAIL and a death domain, such as Fas or an apoptosis-inducing receptor (AI ) Containing, secretory leader sequences described herein can be expressed using the vectors and methods.

Other proteins that can be expressed using the secretory leader sequences, vectors and methods described herein include, for example, clusters of differentiation antigens (referred to as CD proteins), such as Leukocyte Type VI (Proceedings of the
VIth International Works and Conference; edited by Kishimoto, Kikutani et al .; Kobe, Japan, 1996), or CD molecules disclosed in subsequent workshops. Examples of such molecules include CD27, CD30, CD39, CD40; and their ligands (CD27 ligand, CD30 ligand and CD40 ligand). Some of these are members of the TNF receptor family, also including 41BB and OX40; this ligand is often a member of the TNF family (similar to the 4-1BB and OX40 ligands); thus, the TNF and TNFR families The members of can also be expressed using the present invention.

  Enzymatically active proteins may also be expressed using the secretory leader sequences, vectors and methods described herein, and for example, members of the metalloproteinase-disintegrin family, various kinases ( (Including streptokinase and tissue plasminogen activator, and death-related kinases including ankyrin repeats and IKR1 and 2), TNFα converting enzymes, and various other enzymes. Enzymatically active protein ligands can also be expressed by applying the present invention.

  The secretory leader sequences, vectors and methods described herein also include, for example, an immunoglobulin molecule or portion thereof, and a chimeric antibody (an antibody having a human constant region binds to a mouse antigen binding region) or fragment thereof. Useful for the expression of other types of recombinant proteins. Many techniques are known that can manipulate DNA-encoded immunoglobulin molecules to yield DNA that can encode recombinant proteins, such as single chain antibodies, antibodies with increased affinity, or polypeptides based on other antibodies ( See, for example, Larrick et al., Biotechnology 7: 934-938, 1989; Reichman et al., Nature 332: 323-327, 1988; Roberts et al., Nature 328: 731-734, 1987; Verhoeyen et al., Science 239: 1534-1536; And Chaudhary et al., Nature 339: 394-397, 1989).

(Modification at the same time as and after translation)
The present invention can be used during translation or translation by, for example, derivatization with known protecting / blocking groups, proteolytic cleavage, or ligation to antibody molecules or other cellular ligands, such as by glycosylation, acetylation, phosphorylation, amidation. Includes polypeptides that are later differentially modified. Any of numerous chemical modifications include, but are not limited to, cyanogen bromide, trypsin, chymotrypsin, papain, specific chemical cleavage by V8 protease; NaBH _4, acetylation, formylation, oxidation, reduction; and / or It may be performed by known techniques involving metabolic synthesis in the presence of tunicamycin.

  Additional post-translational modifications encompassed by the present invention include, for example, N-linked or O-linked carbohydrate chains (having an N-terminus or C-terminus), attachment of a chemical moiety to the amino acid backbone, N-linked or O-linked Chemical modification of the carbohydrate chain and addition or deletion of the N-terminal methionine residue as a result of prokaryotic host cell expression. Polypeptides may also be modified with a detectable label, eg, an enzyme, fluorescent, isotope or affinity label that allows for the detection and isolation of the protein.

  Also chemically modified derivatives of the polypeptides of the present invention, which may provide additional benefits such as increased solubility, stability and circulation time, or reduced immunogenicity of the polypeptides. Provided by the present invention (see US Pat. No. 4,179,337). The chemical moiety of the derivative may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol and the like. The polypeptide may be modified at random positions within the molecule or at a predetermined position within the molecule and may include 1, 2, 3 or more linked chemical moieties.

  The polymer may be of any molecular weight and may be branched or unbranched. For polyethylene glycol, suitable molecular weights are between about 1 kDa and about 100 kDa for ease of handling and manufacturing (the term “about” is, to some extent, in the preparation of polyethylene glycol. Is larger than the molecular weight mentioned and to some extent smaller). Desired therapeutic profile (eg, desired sustained release period, impact on biological activity, if any, ease of handling, degree or lack of antigenicity, and other polyethylene glycols for therapeutic proteins or analogs) Depending on the known effect) other sizes may be used.

  Polyethylene glycol molecules (or other chemical moieties) must be attached to the protein taking into account its effect on the functional or antigenic domain of the protein. There are a number of conjugation techniques available to those skilled in the art, such as European Patent 0 401 384 (conjugation of PEG to G-CSF); also Malik et al., Exp. Hematol. 20: 1028-1035 (1992) (reporting PEGylation of GM-CSF using tresyl chloride). For example, polyethylene glycol may be covalently linked via an amino acid residue via a reactive group such as a free amino group or a carboxyl group. A reactive group is a group to which an activated polyethylene glycol molecule can be attached. Examples of amino acid residues having a free amino group include lysine residues and N-terminal amino acid residues; residues having a free carboxyl group include aspartic acid residues, glutamine residues and C-terminal amino acid residues. The group can be mentioned. Sulfhydryl groups can also be used as reactive groups to attach polyethylene glycol molecules. Suitable for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.

  One skilled in the art may particularly desire proteins chemically modified at the N-terminus. Using polyethylene glycol as an example of the composition of the present invention, one skilled in the art will be made from various polyethylene glycol molecules (by molecular weight, branching, etc.), the ratio of polyethylene glycol to protein (polypeptide) molecules in the reaction mixture. The type of PEGylation reaction to be performed and the method of obtaining the selected N-terminal PEGylated protein can be selected. The method of obtaining a preparation PEGylated at the N-terminus (ie separating this portion from other monoPEGylated portions as needed) allows for the N-terminal PEGylated material from a population of PEGylated protein molecules. It may be by purification. Selective proteins that have been chemically modified with N-terminal modifications are due to reductive alkylation that elicits different reactivity of different types of primary amino groups (lysine vs. N-terminus) available for derivatization in specific proteins. Can be achieved. Under appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group-containing polymer is achieved.

(Chromosome assay)
In a specific embodiment for chromosome mapping, the cDNA disclosed herein is used to clone the genomic nucleic acid of MGD-CSF. This can be accomplished using various well-known techniques and libraries that are generally commercially available. The genomic DNA is then used for in situ chromosomal mapping using techniques well known for this purpose. Thus, the nucleic acid molecules of the present invention are also useful for chromosome identification. This sequence can be specifically targeted and hybridize to a specific location on an individual human chromosome. In addition, there is currently a need to identify specific sites on the chromosome. Few chromosomal marking reagents are currently available for marking chromosomal locations based on actual sequence data (repetitive polymorphisms). The mapping of DNA to chromosomes according to the present invention is an important first step in associating their sequences with genes associated with disease.

  In short, sequences can be mapped to chromosomes by preparing PCR primers from cDNA. Computer analysis of the 3 'untranslated region is used to rapidly select primers that do not span two or more exons in genomic DNA, which complicates the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.

  PCR mapping of somatic cell hybrids is a rapid procedure for assigning specific DNA to specific chromosomes. Using the present invention with the same oligonucleotide primers, sublocalization can be achieved in a similar manner with a panel of fragments from a particular chromosome or a pool of large genomic clones. Other mapping strategies that can also be used to map chromosomes include in situ hybridization, prescreening with labeled flow-sorted chromosomes and building chromosome-specific cDNA libraries. Preselection by hybridization for the purpose.

Accurate chromosomal location can be obtained in one step using fluorescence in situ hybridization (FISH) of cDNA clones for metaphase chromosome cleavage. This technique can be used with cDNAs as short as about 50-60 bases. For a review of this technology, see Verma et al., Human Chromosomes; A Manual of Basic.
See Techniques, Pergamon Press, New York (1988).

  Once the sequence has been mapped to the exact chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. (Such data can be found, for example, at V. McKusick, Médélian Inheritance in Man available online through the Johns Hopkins University Welch Medical Library.) The relationship between the gene mapped to the same chromosomal region and the disease is then identified through linkage analysis (joint inheritance of physically adjacent genes).

  Differences can then be determined in the cDNA or genomic sequence of affected and unaffected individuals. If the mutation is observed in some or all of the affected individuals, but not in any normal individual, the mutation is considered a causative factor of the disease. At the current resolution of physical mapping and gene mapping techniques, a single cDNA that is precisely located in a chromosomal region associated with disease can be one of 50-500 potential causative genes ( (Assuming 1 megabase mapping resolution and 1 gene per 20 kb).

  The gene encoding MGD-CSF is located on chromosome 16q22.1. Linkage analysis studies suggest that genes on 16q22 are involved in the cause of familial myeloid leukemia (Horwitz et al., Am. J. Hum. Genetics 61: 873-881 (1997)). In this study in a family with 11 related meiotic transmission autosomal dominant acute myeloid leukemias and spinal cord dysplasias, the well-known leukemia metastasis breakpoint regions 21q22.1-q22.2 and 9p22- The linkage to p21 was eliminated. Horwitz et al. Used the microsatellite marker D16S522 to correlate these diseases with a maximum 2-point rod score of 2.82 with the recombinant fraction θ = 0.0, thereby providing linkage evidence for 16q22. Haplotype analysis showed a 16q22 23.5-cM region that was commonly inherited by all affected family members and extended from D16S451 to D16S289. Non-parametric linkage analysis yielded a P-value of 0.00098 for the conditional probability of the linkage. Mutation analysis eliminated the extension of the AT-rich minisatellite repeat FRA16B chromosomal instability and the CAG trinucleotide repeat in the E2F-4 transcription factor present in many growth responsive and growth promoting genes. The “repeat expansion detection” method, which can detect dynamic mutations associated with prediction, more generally eliminated large CAG repeats as a cause of leukemia in this family. MGD-CSF is located on chromosome 16q22.1. Thus, it can potentially play a role in acute myeloid leukemia and spinal cord dysplasia and can be used to treat these diseases.

(Therapeutic compositions and formulations)
The polypeptides, agonists and antagonists of the present invention can be used in combination with a suitable pharmaceutical carrier to include pharmaceutical compositions for parenteral administration. Such compositions comprise a therapeutically effective amount of a polypeptide, agonist or antagonist and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, dextran, water, glycerol, ethanol, and combinations thereof. The formulation must suit the mode of administration.

  The MGD-CSF polypeptide composition takes into account the clinical conditions of the individual subject, the site of delivery of the MGD-CSF polypeptide composition, the method of administration, the schedule of administration, and other factors known to the physician, It is prescribed and administered in a manner consistent with good medical practice. Accordingly, the effective amount of MGD-CSF polypeptide for purposes herein is determined by such considerations.

  The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical composition of the present invention. Accompanying such container (s) may be a precautionary statement in the form prescribed by a government agency that regulates the manufacture, use or sale of a pharmaceutical or biological product. Reflects the approval by the institution of manufacture, use or sale for human administration. Furthermore, the polypeptides, agonists and antagonists of the present invention can be used in combination with other therapeutic compounds.

  The pharmaceutical composition may be administered in a conventional manner, for example, by oral, topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route. The pharmaceutical composition is administered in an amount that is effective for the treatment and / or prevention of the particular indication. Generally, these pharmaceutical compositions are administered in an amount of at least about 10 μg / kg body weight, and in most cases they are administered in a non-excessive amount of about 8 milligrams / kg body weight per day.

  The polypeptides of the present invention, as well as agonist and antagonist compounds that are polypeptides, can also be used according to the present invention by expression of such polypeptides in vivo, ie gene therapy. Thus, for example, a cell may be engineered ex vivo with a polynucleotide (DNA or RNA) encoding a polypeptide; the engineered cell is then provided to a patient. Such methods are well known in the art. For example, cells can be manipulated by procedures known in the art by use of retroviral particles containing RNA encoding the polypeptides of the invention.

  Similarly, cells may be manipulated in vivo to express the polypeptide in vivo, for example, by procedures known in the art. As is known in the art, cells producing retroviral particles containing RNA encoding a polypeptide of the invention are administered to a patient for the purpose of manipulating the cell in vivo and expressing the polypeptide in vivo. May be. These and other methods for administering a polypeptide of the present invention by similar methods should be apparent to those skilled in the art from the teachings of the present invention. For example, the expression vehicle for manipulating the cell may be, for example, an adenovirus other than a retroviral particle that can be used to manipulate the cell in vivo after combination with an appropriate delivery vehicle.

  Retroviruses from which the retroviral plasmid vectors referred to herein above are derived include, but are not limited to, Moloney murine leukemia virus, spleen necrosis virus, rous sarcoma virus, Harvey sarcoma virus, simian leukemia virus, Gibbon leukemia virus, human immunodeficiency virus, adenovirus (HIV), myeloproliferative sarcoma virus and mammary tumor virus.

The nucleic acid sequence encoding the polypeptide of the present invention is under the control of a suitable promoter. The vectors of the present invention contain one or more promoters. Suitable promoters that can be used include, but are not limited to, retroviral long terminal repeats.
repeat) (LTR); SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller et al., Biotechniques, Vol. 9, No. 980-990 (1989), or any other homologous or heterologous Promoters include, but are not limited to, cellular promoters such as eukaryotic cell promoters including histone, pol III and β-actin promoters. Other viral promoters that can be used include, but are not limited to, adenovirus promoters, such as the adenovirus major late promoter; the thymidine kinase (TK) promoter; and the B19 parvovirus promoter.

  Suitable promoters include, but are not limited to, respiratory syncytial virus (RSV) promoter; inducible promoters such as MMT promoter, metallothionein promoter; heat shock promoter; albumin promoter; ApoA1 promoter; human globin promoter; Promoters such as the herpes simplex thymidine kinase promoter; retroviral LTRs (including the modified retroviral LTRs described hereinabove); β-actin promoters; and human growth hormone promoters. The promoter may also be a natural promoter that controls the gene encoding the polypeptide. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.

Retroviral plasmid vectors can be used to transduce packaging cell lines to form producer cell lines. Examples of packaging cells that can be transfected include, but are not limited to, PE501, PA317, PA12, T19-14X, VT-19 as described in Miller, Human Gene Therapy, 1: 5-14 (1990). -17-H2, CRE, CRIP, GP + E-86, GP + envAm12 and DNA cell lines. This vector can transduce packaging cells through any method known in the art. Such methods include, but are not limited to, electroporation, the use of liposomes and CaPO ₄ precipitation. In another method, the retroviral plasmid vector may be encapsulated in liposomes or conjugated to lipids and then administered to the host.

  Producer cell lines produce infectious retroviral vector particles that contain the nucleic acid sequence (s) encoding this polypeptide. Such retroviral vector particles can then be used to transduce eukaryotic cells either in vitro or in vivo. The transduced eukaryotic cell expresses the nucleic acid sequence (s) encoding the polypeptide. Eukaryotic cells that can be transduced include, but are not limited to, embryonic stem cells, embryonal carcinoma cells, and hematopoietic stem cells, hepatocytes, fibroblasts, myeloblasts, keratinocytes, endothelial cells and bronchial epithelial cells. It is done.

  In one embodiment, the MGD-CSF composition is provided in a formulation with pharmaceutically acceptable excipients, a wide variety of which are known in the art (Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th edition (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, seventh Edition, Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, third Edition , Pharmaceutical Press (2000)). Pharmaceutically acceptable excipients such as vehicles, adjuvants, carriers or diluents are publicly available. In addition, pharmaceutically acceptable auxiliary substances such as pH regulators and buffers, toxicity regulators, stabilizers, humectants and the like are publicly available.

  In pharmaceutical dosage forms, the compositions of the invention may be administered in the form of their pharmaceutically acceptable salts, or they may also be alone or in appropriate association, and other pharmaceutically. It may be used in combination with an active compound. The compositions of the invention are formulated according to the potential mode of administration. Factor administration is oral, buccal, nasal, rectal, parenteral, intraperitoneal, intradermal, transdermal, subcutaneous, intravenous, intraarterial, intracardiac, intraventricular, intracranial, intratracheal and subarachnoid It may be achieved by various routes, including intracavitary administration, or otherwise by implantation or inhalation. Accordingly, the compositions of the present invention are formulated into preparations in the form of solids, semisolids, liquids or gases such as tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalations, and aerosols. May be. The following methods and excipients are merely exemplary and are in no way limiting.

  Compositions for oral administration can form solutions, suspensions, tablets, pills, granules, capsules, sustained release formulations, oral rinses or powders. For oral preparations, the factors, polynucleotides and polypeptides can be used alone or with appropriate additives such as conventional additives such as lactose, mannitol, corn starch or potato starch; , Crystalline cellulose, cellulose derivatives, acacia, corn starch, or gelatin; disintegrants such as corn starch, potato starch or sodium carboxymethylcellulose; lubricants such as talc or magnesium stearate; and, if necessary, dilution It may be used in combination with agents, buffering agents, humectants, preservatives and flavoring agents.

  Suitable excipient vehicles are, for example, water, saline, dextran, glycerol, ethanol, and the like, and combinations thereof. In addition, if desired, the vehicle may contain minor amounts of auxiliary substances such as humectants or emulsifiers or pH buffering agents. Practical methods for preparing such dosage forms are known or apparent to those skilled in the art (Gennaro, supra). The composition or formulation to be administered will contain a sufficient amount of factors suitable to achieve the desired condition in the subject being treated.

  The factors, polynucleotides and polypeptides are dissolved, suspended or emulsified in aqueous or non-aqueous solvents such as vegetable oils or other similar oils, synthetic fatty acid glycerides, higher fatty acids or esters of propylene glycol. And, if desired, can be formulated into preparations for injection, together with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizing agents and preservatives. Other formulations for oral or parenteral delivery can also be used as is customary in the art.

  Antibodies, agents, polynucleotides and polypeptides may be utilized in aerosol formulations to be administered via inhalation. The compounds of the present invention can be formulated in pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like. In addition, the agent, polynucleotide or polypeptide composition may be converted into a powder form for nasal or inhalation administration according to the customs of the art.

  In addition, this factor can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. The compositions of the present invention may be administered rectally via a suppository. The suppository may include vehicles such as cocoa butter, carbowaxes and polyethylene glycols which remain solidified at room temperature but melt at body temperature.

  Polynucleotides, polypeptides or other modifiers may also be introduced into tissues or host cells by other routes such as viral infection, microinjection or vesicle fusion. For example, expression vectors can be used to introduce nucleic acid compositions into cells as described above. In addition, jet injection may be used for intramuscular administration (Furth et al., Anal. Biochem. 205: 365-368 (1992)). DNA may be coated on gold particles, and gold microprojectiles are coded with DNA as described in the literature (Tang et al., Nature 356: 152-154 (1992)), It may then be delivered intradermally by a fine particle gun device or “gene gun” that is fired into the skin cells.

  Unit dosage forms for oral or rectal administration such as syrups, elixirs and suspensions may be provided, wherein each dosage unit such as one teaspoon, one tablespoon, one tablet or suppository is provided. A predetermined amount of the composition containing the above factors is included. Similarly, unit dosage forms for injection or intravenous administration may contain the factor (s) in the composition as a solution in sterile water, saline or another pharmaceutically acceptable carrier.

(Identification of factors and antagonists)
The present invention provides modifiers, including polypeptides, polynucleotides and other factors, that increase or decrease the activity of their targets. The modifiers of the invention can act as agonists or antagonists and can interfere with the binding or activity of the polypeptide or polynucleotide. Such modifiers or factors include, for example, agonists or antagonists, polypeptide variants; agonists or antagonists, antibodies; soluble receptors, which are usually antagonists; Small molecule drugs, whether agonists or antagonists; RNAi, which are usually antagonists; antisense molecules, which are usually antagonists; and ribozymes, which are usually antagonists. In certain embodiments, the factor is a polypeptide of the invention, which polypeptide of the invention is itself administered to an individual. In certain embodiments, the agent is an antibody specific for a “target” polypeptide of the invention. In certain embodiments, the agent is a compound, such as a small molecule, that may be useful as an orally available drug. Such modulation includes supplementation with other molecules that directly affect the regulation. For example, an antibody that modulates the activity of a polypeptide of the invention, which is a receptor on the cell surface, can bind to this receptor and immobilize complement, activate the complement cascade, resulting in cell lysis. An agent that modulates the biological activity of a polypeptide or polynucleotide of the invention is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 50, as compared to a suitable control. %, At least about 80%, or at least about 2-fold, at least about 5-fold, or at least about 10-fold or more to increase or decrease activity or binding.

  The invention also provides methods for screening compounds to identify compounds that modulate the biological activity of the polypeptides of the invention. Examples of biological activity of the polypeptides of the invention are described in detail herein, for example in the Examples and Figures.

  The invention further provides a method wherein a mammalian cell or membrane preparation expressing a receptor for a polypeptide of the invention as described above is incubated with a labeled polypeptide of the invention in the presence of the compound. . The ability of the compound to enhance or block this interaction is then measured. Alternatively, the response of a known second messenger system after the interaction of the compound to be screened with the MGD-CSF receptor is measured to determine the ability of this compound to bind to the receptor and elicit a second messenger response And determine whether the compound is a potential agonist or antagonist. Such second messenger systems include, but are not limited to, systems mediated by cAMP, guanylate cyclase, ion channels and phosphoinositide hydrolysis.

  Examples of antagonist compounds include antibodies, or in some cases oligonucleotides, that bind to the receptors of the polypeptides of the invention but do not elicit a second messenger response or MGD-CSF. It binds to the polypeptide itself. Alternatively, a potential antagonist may be a variant of a polypeptide that binds to the receptor but does not elicit a second messenger response and thus effectively blocks the action of the polypeptide.

Another compound that is antagonistic to the MGD-CSF gene and gene product is an antisense construct prepared using antisense technology. Antisense technology may be used to control gene expression through triple helix formation or antisense DNA or RNA; both methods are based on binding of the polynucleotide to DNA or RNA. For example, antisense RNA oligonucleotides of about 10 to about 40 base pairs in length can be designed using the 5 ′ coding portion of the polynucleotide sequence encoding the mature polypeptide of the invention. DNA oligonucleotides are designed to be complementary to a region of a gene involved in transcription, such as a triple helix; Lee et al., Nucl. Acid Res. 6: 3073 (1979); Cooney et al., Science, 241: 456 (1988); and Dervan et al., Science, 251: 1360 (1991); thereby preventing transcription and production of the polypeptides of the invention. . This antisense RNA oligonucleotide hybridizes to mRNA in vivo and is described in Okano, J. et al. Neurochem. , 56: 560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene.
Expression, CRC Press, Boca Raton, Fla. (1988), which blocks translation of mRNA molecules into polypeptides. The above oligonucleotides may also be delivered to cells such that antisense RNA or DNA is expressed in vivo to inhibit polypeptide production.

  Potential antagonist compounds also have a low binding that binds to and occupies the receptor's binding site, thereby rendering the receptor inaccessible to the polypeptide and thereby preventing normal biological activity. Molecule. Examples of small molecules include, but are not limited to, small molecule peptides or peptide-like molecules. Antagonist compounds can be used to inhibit the effects of the polypeptides of the invention, as described in more detail in the Examples and Figures. Antagonists may be used to diagnose, determine prognosis, prevent, and treat immune related diseases, as described in further detail below.

  The present invention also provides methods for identifying factors, such as antibodies, that enhance or block the action of MGD-CSF molecules on cells. For example, these factors can enhance or block the interaction of MGD-CSF binding molecules such as receptors. Factors of interest include both agonists and antagonists. The present invention provides agonists that increase the natural biological function of MGD-CSF and function in a manner similar to MGD-CSF. The invention also provides antagonists that reduce or eliminate the function of MGD-CSF.

  One method of identifying MGD-CSF agonists and antagonists involves biochemical assays after subcellular fractionation. For example, cell compartments, such as membrane or cytoplasmic preparations, can be prepared from cells that express molecules that bind to MGD-CSF molecules, such as signaling or regulatory pathway molecules regulated by MGD-CSF molecules. Subcellular fractionation methods are known in the field of cell biology and may be tailored to produce crude fractions with separate and defined components, such as organelles or organelle membranes. This preparation is incubated with labeled MGD-CSF molecules in the presence or absence of candidate molecules, which may be MGD-CSF agonists or antagonists. The ability of the candidate molecule to interact with the binding molecule or MGD-CSF molecule is reflected in a decrease in the binding of the labeled ligand. Molecules that bind for no reason, i.e., bind without inducing the effects of MGD-CSF molecules, are considered to be mostly antagonists. Molecules that bind well and exert the same or closely related effects as MGD-CSF molecules can potentially prove to be agonists.

  The effects of potential agonists and antagonists are, for example, determining the activity of one or more components of the second messenger system after interaction of the candidate molecule with the cell or appropriate cell preparation, and with this effect It can be measured by comparing the effects of MGD-CSF molecules with those of molecules that exert the same effect as MGD-CSF. Second messenger systems that may be useful in this regard include, but are not limited to, cAMP, cGMP, ion channels, and phosphoinositide hydrolyzed second messenger systems.

  Another example of an assay for the identification of MGD-CSF antagonists is a competitive assay that combines a mixture of MGD-CSF molecules and potential antagonists with membrane bound MGD-CSF receptor molecules. Under suitable conditions for competitive inhibition assays, this assay may also be performed using recombinant MGD-CSF receptor molecules. The MGD-CSF molecule may be labeled, such as by radioactivity, so that the number of MGD-CSF molecules bound to the receptor molecule can be accurately determined to assess the effectiveness of potential antagonists.

  Potential antagonists include small organic molecules, polypeptides and antibodies that bind to a polypeptide of the invention and thereby inhibit or abolish its activity. Potential antagonists may also be present on binding molecules such as receptor molecules that do not induce MGD-CSF-induced activity, thereby preventing the action of MGD-CSF molecules by eliminating MGD-CSF molecules from binding. Small organic molecules, such as closely related proteins or antibodies that bind to the same site, polypeptides. Antagonists of the present invention include fragments of MGD-CSF molecules having the nucleic acid and amino acid sequences shown in the sequence listing.

Other potential antagonists include antisense molecules. Antisense technology can be used, for example, to control gene expression through antisense DNA or RNA, or through triple helix formation. Antisense technology is described, for example, in Okano, J. et al. Neurochem. 56: 560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla (1988). Triple helix formation is described, for example, by Lee et al., Nucleic Acids Research, 6: 3073 (1979); Cooney et al., Science.
241: 456 (1988); and Dervan et al., Science, 251: 1360 (1991). This method is based on the binding of polynucleotides to complementary DNA or RNA. For example, the 5 ′ coding portion of a polynucleotide encoding a mature polypeptide of the invention can be used to design an antisense RNA oligonucleotide of about 10 to about 40 base pairs in length. DNA oligonucleotides are designed to be complementary to regions of the gene involved in transcription, thereby preventing transcription and subsequent production of MGD-CSF molecules. Antisense RNA oligonucleotides hybridize to mRNA in vivo to block translation of the mRNA molecule into MGD-CSF polypeptide. The oligonucleotides described above can also be delivered to cells so that antisense RNA or DNA can be expressed in vivo to inhibit the production of MGD-CSF molecules.

(Diagnosis)
The invention also relates to the use of the genes and gene products of the invention as part of a diagnostic assay for detecting a disease or susceptibility to a disease associated with the presence of a mutation in a nucleic acid sequence encoding a polypeptide of the invention. Individuals carrying mutations in the genes of the present invention can be detected with a DNA label by a variety of techniques. Nucleic acids for diagnosis can be obtained from patient cells, such as from blood, urine, saliva, tissue biopsy and autopsy material. Genomic DNA may be used directly for detection, or by using PCR prior to analysis, for example, as described by Saiki et al., Nature, 324: 163-166 (1986). It may be amplified enzymatically. RNA or cDNA may also be used for the same purpose. As an example, mutants may be identified and analyzed using PCR primers complementary to a nucleic acid encoding a polypeptide of the invention. For example, deletions and insertions can be detected by a change in size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to radiolabeled RNA or radiolabeled antisense DNA sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures.

  Genetic testing based on DNA sequence differences can be accomplished by detecting changes in electrophoretic mobility of DNA fragments during gel electrophoresis in the presence or absence of denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA fragments of different sequences are delayed in their mobility at various positions according to their specific or partial melting points, as described, for example, in Myers et al., Science, 230: 1242 (1985). Can be distinguished on a denaturing formamide gradient gel.

  Sequence changes at specific positions are also described in Cotton et al., Proc. Natl. Acad. Sci. 85: 4397-4401 (1985), as revealed by nuclease protection assays, such as RNase and S1 protection or chemical cleavage methods. Thus, the detection of specific DNA sequences can be performed by hybridization, RNase protection, chemical cleavage, direct DNA sequencing or use of restriction enzymes, such as Restriction Fragment Length Polymorphisms (RFLP) and genomic DNA. Can be achieved by methods such as Southern blotting. In addition to convenient gel electrophoresis and DNA sequencing, mutations may also be detected by in situ analysis.

  The present invention also relates to diagnostic assays for detecting altered levels of MGD-CSF protein in various tissues. Overexpression of these proteins compared to normal control tissue samples can detect abnormal cell proliferation, eg the presence of a tumor. Assays used to detect protein levels in a sample derived from a host are well known to those of skill in the art and include radioimmunoassays, competitive binding assays, Western blot analysis, ELISA assays, “sandwich”. Assays and other assays for expression levels of genes encoding MGD-CSF proteins known in the art are included. Expression can be assayed by qualitatively or quantitatively measuring or evaluating the level of MGD-CSF protein or the level of mRNA encoding MGD-CSF protein in a biological sample. The assay can be performed directly, eg, by detecting or assessing absolute protein or mRNA levels, or by comparing MGD-CSF protein or mRNA to a second biological sample. Can be done. In performing these assays, the MGD-CSF protein or mRNA level in the first biological sample is measured or assessed, and the standard MGD-CSF protein level or mRNA level is measured. Compared to levels; suitable standards include a second biological sample obtained from an individual who does not have the disorder of interest. A standard may be obtained by averaging the level of MGD-CSF in a population of individuals who do not have a disorder associated with MGD-CSF expression. As will be appreciated in the art, once the standard MGD-CSF protein level or mRNA level is known, it can be used repeatedly as a standard for comparison.

  For example, an ELISA assay as described by Coligan et al., Current Protocols in Immunology, 1 (2), Chapter 6 (1991), utilizes antibodies prepared with specificity for the polypeptide antigens of the present invention. In addition, reporter antibodies are prepared against monoclonal antibodies. To the reporter antibody is attached a detection reagent such as a radioactive tag, a fluorescent tag or an enzyme tag, such as horseradish peroxidase. The sample is removed from the host and incubated on a solid support, such as a polystyrene dish, that binds to the protein in the sample. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein, such as bovine serum albumin. A particular antibody, such as a monoclonal antibody, is then incubated in the dish, during which time the antibody binds to any polypeptide of the invention bound to the polystyrene dish. Wash off all unbound monoclonal antibody with buffer. A reporter antibody, eg, a reporter antibody conjugated to horseradish peroxidase, is placed in the dish to cause binding of the reporter antibody to any antibody bound to the protein of interest; unbound reporter antibody is then removed. A substrate, eg, peroxidase, is then added to the dish and present in a given volume of patient sample when compared to a standard by the amount of color that produced the signal, eg, the color developed over a given period of time. The amount of the polypeptide of the present invention is measured.

  A competition assay may be used in which an antibody specific for a polypeptide of the invention is bound to a solid support and labeled MGD-CSF is passed through the solid support with a sample from the host. . The label can be detected and quantified, for example, by liquid scintillation chromatography, and the measurement can be correlated to the amount of the polypeptide of interest present in the sample. A “sandwich” assay may be used in the same manner as an ELISA assay, in which a polypeptide of the invention is passed through a solid support and bound to an antibody module bound to the solid support. To do. A second antibody is then bound to the polypeptide of interest. A third antibody that is labeled and specific for the second antibody then passes through the solid support and is bound to the second antibody. The amount of antibody binding can be quantified; the amount correlates with the amount of polypeptide of interest. See, for example, US Pat. No. 4,376,110.

  The biological samples of the present invention may include any biological sample obtained from a subject, body fluid, cell line, tissue culture or other source that contains MGD-CSF protein or mRNA. As indicated, biological samples include body fluids (eg, serum, plasma, urine, synovial fluid and spinal fluid) containing free MGD-CSF protein, complete or mature MGD-CSF polypeptide or MGD-CSF. Examples include ovarian or renal tissue and other tissue sources that have been found to express the receptor. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. If the biological sample contains mRNA, a tissue biopsy can provide a source.

  Total cellular RNA was obtained from Chomczynski and Sacchi, Anal. Biochem. 162: 156-159 (1987), and can be isolated from biological samples using any suitable technique, such as the single step guanidinium-thiocyanate-phenol-chloroform method. The level of mRNA encoding the MGD-CSF protein can then be assayed using any suitable method. These methods include Northern blot analysis, S1 nuclease mapping, polymerase chain reaction (PCR), reverse transcription combined with polymerase chain reaction (RT-PCR), and reverse transcription combined with ligase chain reaction (RT-LCR). Can be mentioned.

  Total cellular RNA was obtained from Chomczynski and Sacchi, Anal. Biochem. 162: 156-159 (1987), and can be isolated from biological samples using any suitable technique, such as the single step guanidinium-thiocyanate-phenol-chloroform method. The level of mRNA encoding the MGD-CSF protein can then be assayed using any suitable method. These methods include Northern blot analysis, S1 nuclease mapping, PCR, reverse transcription combined with PCR (RT-PCR), and reverse transcription combined with ligase chain reaction (RT-LCR).

  Assaying MGD-CSF protein levels in a biological sample can be performed using antibody-based techniques. For example, MGD-CSF protein expression in tissues can be determined using classical immunohistological methods such as Jalkanen, M et al. Cell. Biol. 101: 976-985 (1985); Jalkanen, M et al., J. Biol. Cell. Biol. 105: 3087-096 (1987). Other antibody-based methods useful for detecting MGD-CSF protein gene expression include immunoassays, such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels such as glucose oxidase, radioisotopes and fluorescent labels such as fluorescein and rhodamine, and biotin.

  In addition to assaying the level of MGD-CSF protein in a biological sample obtained from an individual, MGD-CSF protein may also be detected in vivo by imaging. Antibody labels or markers for in vivo imaging of MGD-CSF protein include those detectable by radiography, NMR or ESR. For radiography, suitable labels include radioactive isotopes, such as barium or cesium, that emit detectable radiation but are not detrimental to the subject. Suitable markers for NMR and ESR include markers with detectable characteristic spins, such as deuterium, which can be incorporated into antibodies by labeling of the relevant hybridoma nutrients.

  An MGD-CSF protein-specific antibody or antibody fragment labeled with a suitable detectable imaging moiety, such as a radioisotope, radiopaque material, or material detectable by nuclear magnetic resonance, is, for example, immune Introduced parenterally, subcutaneously or intraperitoneally to a subject to be tested for systemic disorders. It is understood in the art that the subject used and the size of the imaging system will determine the amount of imaging moiety needed to produce a diagnostic image. The labeled antibody or antibody fragment then accumulates at the location of the cell containing the MGD-CSF protein. In vivo tumor imaging is described in Burchiel et al., Chapter 13, Tumor Imaging: The Radiochemical Detection of Cancer, Masson Publishing, Inc. (1982).

(Therapeutic use of MGD-CSF molecules, agonists and antagonists)
Molecules of the invention and fragments and variants thereof can be used for diagnosis, prognosis, prevention, treatment and treatment of any disorder directly or indirectly mediated by incomplete or insufficient amounts of MGD-CSF. Can be used in development. The MGD-CSF polypeptide, agonist or antagonist can be administered to a patient suffering from such a disorder. Gene therapy approaches can be applied to treat such disorders. The disclosure herein of the sequences of the present invention allows detection of defective MGD-CSF related genes and their replacement with normal or corrected genes. Defective genes can be detected in in vitro diagnostic assays and by comparing the sequences of genes from patients suspected of carrying the defect with the sequences of the present invention.

  Molecules of the invention, such as recombinant MGD-CSF, can have multiple effects on the growth of different cell types and multiple effects on the growth and differentiation of the same cell type under different conditions. Can have. Under conditions where MGD-CSF inhibits proliferation and / or differentiation, recombinant MGD-CSF or related molecules can be used to treat diseases characterized by abnormal proliferation and / or differentiation. Under conditions where MGD-CSF promotes proliferation and / or differentiation, agents that are inhibitory to MGD-CSF or related molecules are used to treat diseases characterized by abnormal proliferation and / or differentiation. obtain. Suitable inhibitors are described herein and can include inhibitory antibodies, small molecule inhibitors, antisense oligonucleotides, siRNA and soluble receptors.

(Disease application)
The molecules of the invention are useful for treating cancer, immune diseases such as autoimmune or inflammatory diseases, ischemic diseases, infectious diseases, bone diseases and neurological diseases. The molecules of the invention are useful for inhibiting the growth of tumor cells or cancer cells and for treating cancer. Thus, the molecules of the invention can be used in a variety of settings for the treatment of animal cancer. Other specific live cancers that can be treated with the molecules of the invention include, but are not limited to, the cancers disclosed below.

  MGD-CSF may play a role in hematopoietic cell retention, proliferation and survival in the bone marrow. Thus, MGD-CSF may be useful in the treatment of hematopoietic cell (eg, neutrophil) defects in cancer patients undergoing chemotherapy or radiation therapy.

  The molecules of the invention can be used to treat lymphoproliferative diseases that result in lymphadenopathy. The molecules of the invention can mediate apoptosis by stimulating clonal deletion of T cells and thus can be used to treat autoimmune diseases and cytotoxic T cell mediated apoptosis that stimulate peripheral tolerance. The molecules of the invention are also used in allergic and systemic lupus erythematosus (SLE), Graves' disease, immunoproliferative lymphadenopathy (IPL), vascular immunoproliferative lymphadenopathy (AIL), immunoblastic lymphadenopathy ( It can be used as a research tool in elucidating the biology of autoimmune disorders including IBL), rheumatoid arthritis, diabetes, and multiple sclerosis, and for treating graft-versus-host disease.

  The molecules of the invention are useful for killing or inhibiting the replication of cells that cause autoimmune or inflammatory diseases, or for treating autoimmune or inflammatory diseases. They can therefore be used in various settings for the treatment of autoimmune or inflammatory diseases in animals.

  The molecules of the invention are also useful for determining, treating, preventing, diagnosing and / or prognosticating diseases including but not limited to autoimmune disorders, immune deficiency disorders and graft-versus-host disease, and recurrent miscarriage It can be. Furthermore, the molecules of the invention can be used as factors to boost immune responsiveness in individuals with transient immune deficiencies. Conditions that produce a temporary immune deficiency that can be ameliorated or treated by administering a molecule of the invention include, but are not limited to, infections such as viral infections (eg, influenza, infectious mononucleosis or measles). Recovery from sexual illness, conditions associated with malnutrition, recovery from or related to stress, recovery from blood transfusion, and recovery from surgery.

  The molecules of the invention can also be used to diagnose, confirm, treat, or prevent the prognosis of one or more of the following diseases, disorders or conditions associated therewith: primary immune deficiency, immune mediated Thrombocytopenia, Kawasaki syndrome, bone marrow transplantation (eg, recent bone marrow transplantation in adults or children), chronic B-cell lymphocytic leukemia, HIV infection (eg, adult or childhood HIV infection), chronic inflammatory demyelinating multiple Neuropathy and posttransfusion purpura.

  In addition, the molecules of the invention can be used to diagnose, confirm, treat or prevent one or more of the following diseases, disorders or conditions associated therewith: Guillain-Barre syndrome, anemia ( For example, anemia associated with parvovirus B19, patients with stable multiple myeloma at high risk of infection (eg, recurrent infection), autoimmune hemolytic anemia (eg, warm autoimmune hemolytic anemia) ), Thrombocytopenia (eg neonatal thrombocytopenia), and immune-mediated neutropenia), transplants (eg, CMV positive organ cytomegalovirus (CMV) -negative recipients), hypogammaglobulinemia (Eg newborns with hypogammaglobulinemia with risk factors for infection or morbidity), epilepsy (eg refractory epilepsy), systemic vascular syndrome, myasthenia gravis (eg For example, myasthenia gravis decompensation), dermatomyositis and polymyositis.

  Additional autoimmune disorders and conditions associated with those disorders that can be treated, prevented, diagnosed and / or confirmed in their prognosis by the molecules of the present invention include, but are not limited to, autoimmune hemolytic anemia , Autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, thread Globe nephritis (eg, IgA nephropathy), multiple sclerosis, neuritis, Buditis ophthalmitis, multiglandular endocrine disorder, purpura (eg, Hennoch-Schönlein purpura), Reiter's disease, stiff man syndrome, self Examples include immune lung inflammation, Guillain-Barre syndrome, insulin-dependent diabetes and autoimmune inflammatory eye diseases.

  Additional autoimmune disorders that can be treated, prevented, diagnosed and / or confirmed in their prognosis by the molecules of the present invention include, but are not limited to, autoimmune thyroiditis; Hashimoto thyroiditis and, for example, cell-mediated Hypothyroidism, including thyroiditis characterized by sexual and humoral thyroid cytotoxicity; SLE (eg, often characterized by circulating and locally generated immune complexes); Goodpasture syndrome (eg, anti-basal) Pemphigus (often characterized by epidermolytic antibodies), eg; receptor autoimmunity, eg Graves' disease (eg, often characterized by antibodies to the thyroid stimulating hormone receptor; Acetylcholine receptor antibody Myasthenia gravis often characterized); insulin resistance (eg often characterized by insulin receptor antibodies); autoimmune hemolytic anemia (eg often characterized by phagocytosis of antibody-sensitive red blood cells); and self Immune thrombocytopenic purpura (for example, often characterized by phagocytosis of antibody-sensitive platelets).

  Additional autoimmune disorders that can be treated, prevented, diagnosed and / or confirmed in their prognosis by the molecules of the present invention include, but are not limited to, rheumatoid arthritis (eg, often by immune complexes in the joints). Scleroderma with anti-collagen antibodies (eg often characterized by antibodies of nucleoli and other nuclei); mixed connective tissue disease (eg extractable nuclear antigens such as ribonucleoproteins) Polymyositis / dermatomyositis (eg, often characterized by non-histone antinuclear antibodies); pernicious anemia (eg, anti-wall cells, anti-microsomes, and anti-intrinsic factor antibodies) Idiopathic Addison's disease (eg, due to humoral and cell-mediated adrenal cytotoxicity) Infertility (eg often characterized by anti-sperm antibodies); Glomerulonephritis (eg often characterized by glomerular basement membrane antibodies or immune complexes); IgA due to primary glomerulonephritis Due to nephropathy; bullous pemphigoid (eg, often characterized by IgG and complement in the basement membrane); Sjogren's syndrome (eg, multiple tissue antibodies and / or specific non-histone antinuclear antibodies (SS Adrenergic agents, including diabetes (eg, often characterized by cell-mediated and humoral islet cell antibodies); and adrenergic resistance with asthma or cystic fibrosis Resistant (eg often characterized by β-adrenergic receptor antibodies Is attached).

  Still further autoimmune disorders whose treatment, prevention, prognosis can be confirmed and / or diagnosed with the antagonist include, but are not limited to, the following disorders: chronic active hepatitis (eg, Primary biliary cirrhosis (eg, often characterized by anti-mitochondrial antibodies); other endocrine insufficiency (eg, in some cases characterized by specific tissue antibodies) Vitiligo (eg, often characterized by anti-melanocyte antibodies); vasculitis (eg, often characterized by complement and / or low serum complement in immunoglobulins and vascular walls); post-myocardial infarction (Eg, often characterized by anti-mitochondrial antibodies); open heart syndrome (Eg often characterized by anti-mitochondrial antibodies); urticaria (eg often characterized by IgG and IgM antibodies to IgE); atopic dermatitis (eg often characterized by IgG and IgM antibodies to IgE) Asthma (often characterized by IgG and IgM antibodies to IgE); inflammatory myopathy; and other inflammation, granulomas, degenerative and atrophic disorders.

  In certain embodiments, molecules of the invention, such as anti-MGD-CSF antibodies, are used to treat or prevent SLE and / or related diseases, disorders or conditions. Lupus-related diseases, disorders and conditions that can be treated or prevented with the molecules of the present invention include, but are not limited to, blood diseases such as hemolytic anemia, leukemia, lymphopenia and thrombocytopenia; immunological disorders Anti-DNA and anti-Sm antibodies, rash, photosensitivity, oral ulcer, arthritis, fever, fatigue, weight loss, serositis, eg pleuritus (pleurisy); kidney disorders, eg nephritis Neurological disorders such as epileptic seizures, peripheral neuropathies and CNS related disorders; gastrointestinal disorders; Raynaud's phenomenon; and epicarditis.

  The molecules of the invention can also be used to inhibit neoplasia such as tumor cell growth. MGD-CSF polypeptides can be responsible for tumor destruction through apoptosis and cytotoxicity against specific cells. Diseases associated with increased cell viability or inhibition of apoptosis that can be treated, prevented, diagnosed and / or confirmed in prognosis by the molecules of the present invention include, but are not limited to, cancers (eg, Follicular lymphoma, carcinoma with p53 mutation and hormone-dependent tumor, including but not limited to colon cancer, heart tumor, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestine Cancer, testicular cancer, gastric cancer, neuroblastoma, myoma, myoma, lymphoma, endothelial tumor, osteoblastoma, giant cell tumor, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi sarcoma and ovarian cancer Autoimmune disorders (eg, multiple sclerosis, Sjogren's syndrome, Graves' disease, Hashimoto's thyroiditis, autoimmune diabetes, biliary cirrhosis, Beechette's disease, Crohn's disease, polymyositis, all Lupus erythematosus and immune-related glomerulonephritis, autoimmune gastritis, autoimmune thrombocytopenic purpura and rheumatoid arthritis) and viral infections (eg, herpesviruses, poxviruses and adenoviruses), inflammation, graft-versus-host disease (acute and And / or chronic), acute graft rejection, and chronic graft rejection. In certain embodiments, the present invention is used to inhibit the growth, progression and / or metastasis of cancer, in particular cancer as described above or in the following paragraphs.

  Additional diseases or conditions associated with increased cell viability that can be treated, prevented, diagnosed and / or confirmed in accordance with the present invention include, but are not limited to, malignant tumors and related disorders. E.g. leukemia (including acute leukemia (e.g. acute lymphoblastic leukemia, acute myeloid leukemia, including myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia)), And chronic leukemia (eg, chronic myelogenous (granulocytic) leukemia and chronic lymphocytic leukemia), myelodysplastic syndrome, polycythemia vera, lymphoma (eg, Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease and solid tumors, including but not limited to sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, soft Sarcoma, osteogenic sarcoma, chordoma, hemangiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovial tumor, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma Pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell cancer, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary cancer, papillary gland cancer, cystadenocarcinoma, medullary cancer, primary bronchial cancer, Renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonic cancer, Wilms tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocyte Cytoma, medulloblastoma, craniopharyngioma, ventricular ependymoma, pineal gland, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblast And retinoblastoma Progression and / or metastasis of 瘍 thereof.

  Diseases associated with increased apoptosis that can be treated, prevented, diagnosed and / or confirmed in prognosis by the molecules of the present invention include, but are not limited to, AIDS (eg, HIV-induced neuropathy). And HIV encephalitis), neurodegenerative disorders (eg, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration and brain tumors or previously related diseases), autoimmune disorders, eg, multiple Sclerosis, Sjogren's syndrome, Graves' disease, Hashimoto's thyroiditis, autoimmune diabetes, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus, immune-related glomerulonephritis, autoimmune gastritis , Thrombocytopenic purpura, and rheumatoid arthritis, myelodysplastic syndromes (eg, Anemia), graft-versus-host disease (acute and / or chronic), ischemic injury (such as caused by myocardial infarction, stroke and reperfusion injury), liver injury or disease (eg, hepatitis-related liver injury, cirrhosis, Ischemia / reperfusion injury, cholestasis (bile duct injury) and liver cancer), toxic-induced liver disease (such as caused by alcohol), septic shock, ulcerative colitis, cachexia and Anorexia may be mentioned.

  Another embodiment of the invention relates to the use of MGD-CSF polynucleotides, polypeptides or antagonists to reduce TGD MGD-CSF or NP_686969 mediated death in HIV infected patients. The role of T cell apoptosis in AIDS development has been the subject of numerous studies (eg, Meyaard et al., Science, 257: 217-219 (1992); Groux et al., J. Exp. Med., 175: 331 (1992)). And Oyaizu et al., Cell Activation and Apoptosis in HIV Infection, edited by Andrieu and Lu, Plenum Press, New York, pages 101-114 (1995)).

  Apoptosis of T cells appears to occur through a number of mechanisms. Fas-mediated apoptosis has been implicated in T cell loss in HIV individuals (Katsikis et al., J. Exp. Med. 181: 2029-2036 (1995). Activated human T cells are induced to activate. Upon induction through the CD3 / T cell receptor complex, a process termed activated-induced cell death (AICD), it is induced to undergo programmed cell death (apoptosis). AICD of CD4 T cells isolated from asymptomatic individuals infected with HIV has been reported (Groux et al., Supra) Thus, ACID plays a role in CD4 + T cell depletion and progression to AIDS in HIV infected individuals Thus, the present invention provides MGD-CS in HIV patients. There is provided a method of inhibiting F-mediated T cell death, comprising the step of administering to the patient a molecule of the invention, wherein in one embodiment, the patient is an MGD-CSF polynucleotide, Asymptomatic when treatment with the polypeptide or antagonist begins: Optionally, prior to treatment, peripheral blood T cells are extracted from HIV patients and MGD-CSF mediated by procedures known in the art. In one embodiment, the patient's blood or plasma is contacted ex vivo with a molecule of the invention, such as an anti-MGD-CSF or NP_686969 antibody, wherein the antibody or other antagonist is It can be bound to a suitable chromatography matrix by procedures known in the art. Flow through a chromatography column containing the bound antagonist trix, it is then returned to the patient. Fixed antagonist binds to MGD-CSF or NP_689699, thus they are removed from the patient's blood.

  In further embodiments, the molecules of the invention are administered in combination with other inhibitors of T cell apoptosis. For example, as noted above, Fas-mediated apoptosis is also involved in T cell depletion in HIV positive individuals (Katsikis et al., J. Exp. Med., 181: 2029-2036 (1995)). Thus, patients susceptible to both Fas ligand-mediated and MGD-CSF-mediated T cell death may have factors that block the interaction of MGD-CSF or NP_669999 with their receptors, and Fas ligand / Fas interaction. It can be treated with both blocking factors. Suitable factors for blocking Fas ligand binding to Fas include, but are not limited to, soluble Fas polypeptides; multimeric forms of soluble Fas polypeptides (eg, dimers of sFas / Fc); organisms that cause apoptosis Anti-Fas antibody that binds to Fas without inducing a biological signal; an anti-Fas ligand antibody that blocks the binding of Fas ligand to Fas; and a Fas that binds to Fas but does not induce a biological signal that results in apoptosis Examples include ligand muteins (muteins). Monoclonal antibodies can be used in accordance with the present invention. Examples of factors suitable for blocking Fas ligand / Fas interactions, including blocking anti-Fas monoclonal antibodies, are described in WO95 / 10540.

  In another example, an agent that blocks the binding of MGD-CSF or NP_6899699 to the receptor is administered with a molecule of the invention. Such factors include, but are not limited to, soluble MGD-CSF receptor polypeptide, multimeric forms of soluble receptor polypeptide, and bind to MGD-CSF or NP — 699699 receptor without inducing biological signals that cause apoptosis. MGD-CSF receptor antibodies, antibodies that block the binding of MGD-CSF or NP_689699 to one or more receptors, and muteins that bind to the receptor but do not induce biological signals that cause apoptosis .

  The molecules of the invention can also be used to regulate hematopoiesis, including erythropoiesis. Hematopoiesis is a multi-step process of cell growth and differentiation that begins with a pool of pluripotent stem cells. These cells can proliferate and differentiate into hematopoietic progenitors in response to various stimuli. The molecules of the invention can be used to stimulate or inhibit the development of hematopoietic cells, eg, erythropoietic cells.

  In certain embodiments, the molecules of the invention are used to treat or prevent bone diseases. The molecules of the present invention promote the differentiation of hematopoietic stem cells into osteoclast precursor cells. Thus, the molecules of the invention can be used to treat bone diseases such as those characterized by defects in osteoclast differentiation and function, such as osteoporosis. MGD-CSF and related molecules can be used as therapeutic agents, eg, protein therapeutic agents, or in gene therapy to treat these diseases.

  In certain embodiments, the molecules of the invention are used to treat or prevent neurological diseases. The molecules of the present invention promote the differentiation of hematopoietic stem cells into microglia progenitor cells. Accordingly, the molecules of the present invention are useful for diseases such as neurological diseases characterized by defects in microglia differentiation and function, such as Alzheimer's disease, multiple sclerosis, acute disseminated cerebrospinal disorder, progressive multifocal leukoencephalopathy. Can be used to treat, stroke, and Parkinson's disease. MGD-CSF and related molecules can be used as therapeutic agents to treat these diseases, for example as protein therapeutics or in gene therapy.

  In certain embodiments, the molecules of the invention are used to treat or prevent cardiovascular disorders, including peripheral arterial diseases such as limb ischemia. Cardiovascular disorders include cardiovascular abnormalities such as arterio-arterial fistula, arteriovenous fistula, cerebral arteriovenous malformation, congenital heart defects, pulmonary atresia and scimitar syndrome. Congenital heart defects include aortic constriction, trilobular heart, coronary vessel anomalies, crisscross heart, right thoracic heart, patent ductus arteriosus, Ebstein Malformation, Eisenmenger complex, left ventricular dysgenesis syndrome, left thoracic heart, trilogy of Fallot, tetralogy of Fallot, macrovascular transposition, bilateral major right ventricular origin, tricuspid atresia, arterial canal Residual and heart septal defects, such as aortopulmonary septal defect, endocardial floor defect, Rutambasche syndrome, and ventricular heart septal defect ects) and the like.

  Cardiovascular disorders that can be treated with the molecules of the invention also include heart diseases such as arrhythmia, carcinoid heart disease, high cardio output, low cardio output, cardiac tamponade , Endocarditis (including bacterial), cardiac aneurysm, cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, cardiac hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricle Hypertrophy, post-infarct heart rupture, ventricular septal rupture, heart valve disease, myocardial disease, myocardial ischemia, endocardial fluid effusion, epicarditis (including infarct and tuberculosis), Intrapericardial emphysema, postpericardiotomy syndrome, right heart disease, rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy complication (cardiovascular) regnancy complications), Scimitar Syndrome, cardiovascular syphilis, and cardiovascular tuberculosis (Cardiovascular tuberculosis) and the like.

  Arrhythmias that can be treated using the molecules of the present invention include sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia, extrasystole, Adams-Stokes syndrome, leg block, sinoatrial block, long QT syndrome (long) QT syndrome, minor contractions, Lone-Gannong-Levine syndrome, Mahaim-type pre-excitation syndrome, Wolf-Parkinson-White syndrome, sinus insufficiency syndrome, tachycardia, and ventricular fibrillation It is done. Tachycardia that can be treated with the molecules of the present invention include paroxysmal tachycardia, supraventricular tachycardia, ventricular intrinsic rhythm promotion, atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia Ectopic junction tachycardia, sinoatrial nodal reentry tachycardia, sinus tachycardia, torsade de pointe, and ventricular tachycardia. Heart valve diseases include aortic dysfunction, aortic stenosis, heart murmurs, aortic valve prolapse, mitral valve prolapse, tricuspid prolapse, mitral valve dysfunction, mitral valve Examples include stenosis, pulmonary valve atresia, pulmonary valve dysfunction, pulmonary valve stenosis, tricuspid atresia, tricuspid valve dysfunction, and tricuspid stenosis.

  Myocardial diseases that can be treated with the molecules of the present invention include alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, valvular aortic stenosis, valvular pulmonary stenosis, restrictive cardiomyopathy, Chagas Cardiomyopathy, endocardial fibroelastosis, endocardial myocardial fibrosis, Keynes syndrome, myocardial reperfusion injury, and myocarditis. Myocardial ischemia that can be treated with the molecules of the present invention includes coronary artery disease such as angina, coronary aneurysm, coronary atherosclerosis, coronary artery thrombosis, coronary vasospasm, myocardial infarction, and myocardial stunning. It is done.

  Cardiovascular diseases that can be treated with the molecules of the present invention also include vascular diseases such as aneurysms, angiogenesis abnormalities, hemangiomas, bacterial hemangiomas, Hippel-Lindau disease, Klipper- Tornonay-Weber syndrome, Sturge-Weber syndrome, vasomotor edema, aortic disease, Takayasu arteritis, aortitis, Lleish syndrome, arterial occlusion disease, arteritis, arteritis, nodular polyarteritis , Cerebrovascular disorder, diabetic vascular disorder, diabetic retinopathy, embolism, thrombosis, acromegaly, sputum, hepatic vein occlusion disorder, hypertension, hypotension, ischemia, peripheral vascular disorder, phlebitis, pulmonary veins Obstructive disease, Raynaud's disease, CREST syndrome, retinal vein occlusion, scimitar syndrome, superior vena cava syndrome, telangiectasia, vasodilatation Sex ataxia (atacia telangiectasia), hereditary hemorrhagic telangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis, and venous insufficiency and the like. Aneurysms include dissecting aneurysms, pseudoaneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, cardiac aneurysms, and iliac aneurysms .

  Arterial occlusive diseases that can be treated with the molecules of the present invention include arteriosclerosis, intermittent claudication, carotid stenosis, fibromuscular dysplasia, mesenteric vascular occlusion, moyamoya disease, renal artery occlusion, retinal artery occlusion And occlusive thrombotic vasculitis.

  Cerebrovascular disorders that can be treated with the molecules of the present invention include carotid artery disease, cerebral amyloid vascular disorder, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous congenital anomalies, cerebral artery disease, cerebral embolism And thrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg syndrome, intracerebral hemorrhage, epidural hematoma, subdural hematoma, subarachnoid hemorrhage, cerebral infarction, cerebral ischemia (transient ), Subclavian artery steal syndrome, periventricular leukomalacia, vascular headache, cluster headache, migraine, and vertebrobasilar dysfunction.

  Embolisms that can be treated with the molecules of the present invention include air embolism, amniotic fluid embolism, cholesterol embolism, toe cyanosis syndrome, fat embolism, pulmonary embolism, and thromboembolism. Thrombosis includes coronary artery thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid artery thrombosis, sinus thrombosis, Wallenberg syndrome, and thrombophlebitis.

  Ischemia that can be treated with the molecules of the present invention include cerebral ischemia, ischemic colitis, compartment syndrome, anterior compartment syndrome, myocardial ischemia, reperfusion injury, and peripheral limb imagination. Blood. Vasculitis that can be treated with the molecules of the present invention includes aortitis, arteritis, Behcet syndrome, Churg-Strauss syndrome, mucocutaneous lymph node syndrome, obstructive thrombotic vasculitis, hypersensitivity vasculitis, Shane Examples include Schoenlein-Henoch purpura, allergic dermatovasculitis and Wegener's granulomatosis.

  The present invention further provides for the treatment of diseases associated with neovascularization by administration of the molecules of the present invention. Malignant and metastatic conditions that can be treated with the molecules of the present invention include, but are not limited to, malignant tumors, solid tumors and cancers described herein, otherwise known in the art (this For a review of such disorders, see Fishman et al., Medicine, 4th edition, JB Lippincott Co., Philadelphia (1997)).

  Further, ophthalmic disorders associated with neovascularization that can be treated with the molecules of the present invention include, but are not limited to, neovascular glaucoma, diabetic retinopathy, retinoblastoma, post-lens fibroplasia, uveitis, immaturity Retinopathy, macular degeneration, corneal transplant neovascularization, and other ocular inflammatory diseases, ocular tumors, and diseases associated with neovascularization of the choroid or iris. For example, Waltman et al., Am. J. et al. Ophthal. 85: 704-710 (1978) and Gartner et al., Surv. Ophthal. 22: 291- 312 (1978).

  Further, disorders that can be treated with the molecules of the present invention include, but are not limited to, hemangiomas, arthritis, psoriasis, hemangiofibromas, atheromatous plaques, delayed wound healing, granulation, hemophilic joints, hyperplasia Include spastic scars, false joint fractures, Osler-Weber syndrome, purulent granulomas, scleroderma, trachoma, and vascular adhesions.

  Molecules and antagonists thereof of the present invention include, but are not limited to, cancer (eg, immune cell related cancer, breast cancer, prostate cancer, ovarian cancer, follicular lymphoma, cancer with p53 mutation or modification, brain tumor, bladder cancer, cervical Useful in the diagnosis and treatment or prevention of a wide variety of diseases and / or conditions, including cancers of the colon, colon, colorectal, non-small cell lung, and small cell carcinoma of the lung, gastric cancer, and the like. They may also include lymphoproliferative disorders (eg, lymphadenopathy), microbial disorders (eg, viruses, bacteria, etc.), infections such as HIV-1 infection, HIV-2 infection, herpes virus infection (but not limited to HSV). -1, HSV-2, CMV, VZV, HHV-6, HHV-7, EBV), adenovirus infection, poxvirus infection, human papilloma virus infection, hepatitis infection (eg, HAV, HBV, HCV, etc.) ), Helicobacter pylori infection, invasive Staphylococci, etc.) and parasitic infections are useful in diagnosis and treatment or prevention. They further include nephritis, bone disease (eg, osteoporosis), atherosclerosis, pain, cardiovascular disorders (eg, neovascularization, reduced neovascularization), and decreased circulation (eg, ischemic disease, eg, Myocardial infarction, stroke, etc. AIDS, allergy, inflammation, neurodegenerative copyright (eg Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration, transplant rejection (acute and chronic), Graft-versus-host disease, diseases arising from bone and spinal cord dysplasia (eg, aplastic anemia), rheumatoid joint tissue destruction, liver disease (eg, acute and chronic hepatitis, liver damage, biliary tract disease, and cirrhosis), self Immune diseases (eg, multiple sclerosis, rheumatoid arthritis, SLE, immune complex glomerulonephritis, autoimmune diabetes, autoimmune thrombocytopenic purpura, Graves' disease, bridge Thyroiditis, etc.), cardiomyopathy (eg dilated cardiomyopathy), diabetes, diabetic complications (eg diabetic nephropathy, diabetic neuropathy, diabetic retinopathy), influenza, asthma, psoriasis, glomeruli Useful in the diagnosis and treatment or prevention of nephritis, septic shock and ulcerative colitis.

  The molecules of the invention are useful for promoting angiogenesis and wound healing (eg, wounds, burns and fractures). They are also useful as adjuvants that enhance immune responsiveness to specific antigens and / or antiviral immune responses.

  More particularly, the molecules of the invention are useful for modulating immune responses. For example, they may be useful in preparing for or recovering from surgery, trauma, radiation therapy, chemotherapy and transplantation, and to boost the immune response and / or recovery process in the elderly and immunocompromised individuals Can be used. They are useful, for example, as immunosuppressants in the treatment or prevention of autoimmune disorders. In certain embodiments, the molecules of the invention are used to treat or prevent chronic inflammation, allergies or autoimmune conditions as described herein above or otherwise known in the art. Used.

  Uses of the molecules of the present invention include, but are not limited to, adult respiratory distress syndrome, anaphylaxis, allergic asthma, allergen rhinitis, drug allergy (eg to penicillin or cephalosporin), primary central neurolymphoma (PCNSL), glia Blastoma, chronic lymphocytic leukemia (CLL), lymphadenopathy, rheumatoid arthritis, osteoarthritis, acute lymphoblastic leukemia (ALL), Hodgkin's disease and non-Hodgkin's disease lymphoma, eye disease, uveoretinitis, Treatment or prevention of autoimmune stages of type 1 diabetes, myasthenia gravis, autoimmune liver damage, autoimmune inflammatory bowel disease and Crohn's disease. A combination of MGD-CSF protein and an immunotherapeutic agent, such as IL-2 or IL-12, can produce a synergistic or additive effect useful in treating established cancer.

  Furthermore, the molecules of the invention can be used not only as therapeutic molecules as described herein, but also as search tools in elucidating the biology of tumor-related diseases such as cancer. Thus, the molecules of the invention are useful for inhibiting the manipulation of tumor cells or cancer cells, or for treating cancer in animals. Thus, the molecules of the invention can be used in a variety of settings for the treatment of animal cancers such as sarcomas, adenomas, adenocarcinomas, carcinomas, papillomas, lymphomas and the like. Other specific types of cancer that can be treated with the molecules of the invention include, but are not limited to, prostate cancer, breast cancer (including, for example, intraductal and inflammatory), colon cancer, colorectal cancer, bladder cancer, ovary Examples include cancer, cervical cancer, papillary cancer, papillary adenocarcinoma, cystadenocarcinoma, embryonal carcinoma, uterine cancer and testicular cancer.

(Antibodies and vaccines)
(antibody)
Antibodies specific for MGD-CSF or NP_689669 are suitable for use in the present invention and can be raised against an intact MGD-CSF protein or antigenic polypeptide fragment thereof. This protein or fragment may be given to animals such as rabbits or mice with or without a carrier protein such as albumin. In general, polypeptide fragments are sufficiently immunogenic to produce a satisfactory immune response without a carrier if they are at least about 25 amino acids in length.

The antibodies of the present invention include polyclonal and monoclonal antibody preparations, as well as hybrid antibodies, modified antibodies, preparations comprising chimeric and humanized antibodies, and hybrid (chimeric) antibody molecules (eg, Winter et al., Nature). 349: 293-299 (1991)); and US Pat. No. 4,816,567); F (ab ′) ₂ , and F (ab) fragments; Fv molecules (noncovalent heterodimers, See, e.g., Inbar et al., Proc. Natl. Acad. Sci. 69: 2659-1662 (1972)); and Ehrlich et al. (1980) Biochem 19: 4091-4096); single chain Fv molecules (sFv) (e.g. Huston et al., Proc. Natl. Acad. ci.85: 5879-5883 (1980); dimeric and trimeric antibody fragment constructs; minibodies (eg, Pack et al., Biochem. 31: 1579-1584 (1992); Cumber et al., J. Immunology 149B: 120-126 (1992)); humanized antibody molecules (eg, Riechmann et al., Nature 332: 323-327 (1988); Verhoeyan et al., Science 239: 1534-1536 ( 1988); heteroconjugated antibodies and bivalent specific antibodies (see, eg, US Pat. No. 6,010,902 and US Patent Application No. 2002/0155604); and such From a simple molecule A functional fragment of interest, wherein such a fragment retains specific binding.

  Methods for making monoclonal and polyclonal antibodies are known in the art. A monoclonal antibody is generally an antibody having a homologous antibody population. The term is not limited regarding the species or source of the antibody, nor is it limited by the manner in which it is made. The term encompasses whole immunoglobulins. Polyclonal antibodies are produced by immunizing a suitable animal such as a mouse, rat, rabbit, sheep or goat with an antigen of interest such as a stem cell transformed with a gene encoding the antigen. In order to enhance immunogenicity, the antigen may be combined with a carrier prior to immunization. Suitable carriers are typically large slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, lipid aggregates (eg oil droplets or liposomes) and It is an inactive virus particle. Such carriers are well known to those skilled in the art. Furthermore, the antigen may be conjugated to a bacterial toxin such as a toxin derived from diphtheria, tetanus, cholera, etc. to enhance its immunogenicity.

  In addition, a technique developed for the production of chimeric antibodies (Morrison et al.) By splicing a gene from an appropriate antigen-specific mouse antibody molecule together with a gene from an appropriate biologically active human antibody molecule. Proc. Natl. Acad. Sci., 81: 851-855 (1984); Neuberger et al., Nature, 312; 604-608 (1984); Takeda et al., Nature, 314: 452-454 (1985)). Also good. Chimeric antibodies, where different portions are antibodies from different animal species, eg, antibodies having variable regions from human monoclonal antibodies and human immunoglobulin constant regions, eg, humanized antibodies, and insertion / deletion for cdr and framework regions Antibodies with loss are suitable for use in the present invention.

  The invention also encompasses humanized antibodies, ie, antibodies that are mostly human immunoglobulin sequences. Humanized antibodies of the present invention generally refer to non-human immunoglobulin that has been modified to incorporate portions of human sequences. A humanized antibody can include a human antibody comprising the entire human immunoglobulin sequence.

  The antibodies of the present invention can be prepared by any of a variety of methods. For example, cells expressing MGD-CSF or NP-689669 protein or an antigenic fragment thereof may be administered to an animal to induce production of serum containing polyclonal antibodies. The preparation of MGD-CSF or NP_6869669 protein is prepared and purified so as to be substantially free of natural contamination, and the preparation can be introduced into animals to produce polyclonal antisera with specific binding activity.

  The antibodies of the invention specifically bind to their respective antigen (s); they have a high binding activity and / or a high affinity for a particular polypeptide or, more precisely, for an epitope of an antigen. May show gender. An antibody of the invention may bind to one epitope or to more than one epitope. They may exhibit different affinity and / or binding activity for different epitopes on one or more molecules. If an antibody binds more strongly to one epitope than to another epitope, the result of modulation of the binding conditions is that, in some cases, it binds almost exclusively to this particular epitope, and the same polypeptide It does not bind to any other epitope above and cannot bind to polypeptides that do not contain this epitope.

  The invention also provides monoclonal antibodies and MGD-CSF or NP_686969 protein binding fragments thereof. Monoclonal antibodies of the present invention can be obtained using hybridoma technology, such as Kohler et al., Nature 256: 495 (1975); Kohler et al., Eur. J. et al. Immunol. 6: 511 (1976); Kohler et al., Eur. J. et al. Immunol. 6: 292 (1976); Hammerling et al .: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N .; Y. (1981) 563-681. In general, such a procedure involves immunizing an animal (eg, a mouse) with MGD-CSF protein antigen, or MGD-CSF protein expressing cells. Appropriate cells can be recognized by their ability to bind anti-MGD-CSF protein antibodies. Such cells are in any suitable tissue culture medium; for example supplemented with 10% fetal calf serum (inactivated at about 56 ° C.) and about 10 g / l non-essential amino acid, about 1,000 U. May be cultured in Earle's modified Eagle's medium supplemented with / ml penicillin and about 100 μg / ml streptomycin. Such mouse splenocytes are extracted and fused with an appropriate myeloma cell line. Any suitable myeloma cell line can be used in accordance with the present invention; for example, the parent myeloma cell line (SP20) available from the American Type Culture Collection (ATCC), Manassas, Va. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium and then cloned by limiting dilution as described, for example, by Wands et al. (Gastroenterology 80: 225-232 (1981)).

  Alternatively, antibodies that can bind to MGD-CSF or NP_686969 protein antigens can be generated in a two-step procedure through the use of anti-idiotype antibodies. Such a method takes advantage of the fact that the antibody is itself an antigen and thus it is possible to obtain an antibody that binds to the second antibody. According to this method, an animal such as a mouse is immunized with a specific antibody. The spleen cells of such animals are then used to produce hybridoma cells, and the hybridoma cells are screened to produce a clone that produces an antibody whose ability to bind to a specific antibody can be blocked by the antigen. Is identified. Such antibodies include anti-idiotypic antibodies against antibodies specific for MGD-CSF or NP_686969 protein and can be used to immunize animals to induce the formation of additional specific antibodies.

It will be appreciated that Fab and F (ab ′) ₂ and other fragments of the antibodies of the present invention may be used in accordance with the methods disclosed herein. Such fragments are typically produced by proteolytic cleavage using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F (ab ′) ₂ fragments). Alternatively, MGD-CSF protein binding fragments can be generated through the application of recombinant DNA technology or through synthetic chemistry. Humanized chimeric monoclonal antibodies are suitable for in vivo use of anti-MGD-CSF in humans. Such humanized antibodies can be produced using genetic constructs derived from hybridoma cells that produce the monoclonal antibodies described above. Methods for producing chimeric antibodies are known in the art. For reviews, see Morrison, Science 229: 1202 (1985); Oi et al., BioTechniques 4: 214 (1986); Cabilly et al., US Pat. No. 4,816,567; See Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne et al., Nature 312: 643 (1984); Neuberger et al., Nature 314: 268 (1985).

(vaccine)
The present invention provides a method for such treatment of a subject in need of or desired prophylactic or therapeutic treatment by providing a vaccine that can be administered to the subject. The present invention elicits an immune response against a subject by providing a substantially purified polypeptide or active fragment derived from SEQ ID NOs: 7-12; provides a vaccine composition, and the polypeptide and vaccine Also provided are methods for administering the composition to a subject. This vaccine is useful for treating cancer, proliferative, inflammatory, immune, metabolic, bacterial or viral disorders, one or more polynucleotides, polypeptides or modifiers of the invention, such as antibodies It may comprise a vaccine composition, a polypeptide vaccine composition or a polynucleotide vaccine composition.

  For example, the vaccine may be a cancer vaccine and the polypeptide may be a cancer antigen at the same time. The vaccine may be an anti-inflammatory vaccine and the polypeptide may simultaneously be an inflammation-related antigen. The vaccine may be a viral vaccine and the polypeptide may simultaneously be a viral antigen. In certain embodiments, the vaccine comprises a polypeptide fragment, the fragment comprising at least one extracellular fragment of a polypeptide of the invention and / or of a signal peptide from at least one extracellular fragment of a polypeptide of the invention. Those not present are included for the treatment of proliferative disorders such as cancer. In certain embodiments, the vaccine comprises a polynucleotide encoding one or more such fragments that are administered for the treatment of a proliferative disorder, such as, for example, cancer. Further, the vaccine may be administered with or without an adjuvant. The vaccine may be administered with the polypeptides shown in the tables and sequence listings; it may be administered prior to, substantially simultaneously with, or after administering the polypeptides.

  Vaccine therapy involves the use of a polynucleotide, polypeptide or factor of the invention as an immunogen for tumor antigens (Machiels et al., Semin. Oncol. 29: 494-502, 2002). For example, a vaccine based on a peptide of the invention comprises an unmodified polypeptide of the invention, a fragment thereof, and an MHC class I and class II restriction peptide (Knutsonra, J. Clin. Invest. 07: 477-484, 2001). Including, for example, disclosed sequences having universal, non-specific MHC class II restricted epitopes. Vaccines based on peptides containing tumor antigens may be given directly, alone or in combination with other molecules. The vaccine may also be delivered orally by producing the antigen in a transgenic plant, which can then be taken orally (US Pat. No. 6,395,964).

  In certain embodiments, the antibody itself can be used as an antigen in an anti-idiotype vaccine. That is, by administering an antibody against a tumor antigen, B cells are stimulated to produce an antibody against that antibody, which in turn recognizes tumor cells.

  Nucleic acid-based vaccines can deliver tumor antigens as polynucleotide constructs that encode the antigen. Vaccines containing genetic material such as DNA or RNA may be administered directly, alone or in combination with other molecules. Administration of a vaccine expressing a molecule of the invention results in sustained expression and release of the therapeutic immunogen over time, helping to control unwanted tumor growth, eg, as plasmid DNA.

  In certain embodiments, the nucleic acid based vaccine encodes an antibody of the invention. In such embodiments, the vaccine (eg, a DNA vaccine) may include a post-transcriptional regulatory element, eg, a post-transcriptional regulatory RNA element (WPRE) from Woodchuck Hepatitis Virus. These post-transcriptional regulatory elements can be used against the tumor microenvironment to target antibodies, or fusion proteins comprising antibodies and costimulatory molecules (Pertl et al., Blood, 101: 649-654, 2003). .

In addition to stimulating an anti-tumor immune response by inducing a humoral response, the vaccines of the present invention may also induce a cellular response that induces T cell stimulation that directly recognizes and kills tumor cells. For example, the nucleotide-based vaccines of the invention that encode tumor antigens can be used to activate the arms of the immune system's CD8 ⁺ cytotoxic T lymphocytes.

  In certain embodiments, the vaccine activates T cells directly, while the antigen-presenting cells support T cell activation. Killer T cells are primed in part by interaction with antigen presenting cells, such as dendritic cells. In certain embodiments, a plasmid comprising a nucleic acid molecule of the invention enters an antigen presenting cell, which in turn presents the encoded tumor antigen, which contributes to killer T cell activation. Again, tumor antigens can be delivered as plasmid DNA constructs alone or with other molecules.

  Since MGD-CSF and NP_689669 can promote in vitro dendritic cell differentiation from either human bone marrow CD34 + stem cells or peripheral blood monocytes, the molecules of the invention can be used to expand dendritic cells ex vivo. Can be. The expanded cell population may then be returned to the patient, for example, as a dendritic cell vaccine. Furthermore, the molecules of the present invention may promote dendritic cell differentiation from autologous hematopoietic stem cells and / or monocytes in vivo in patients, which enhances the patient's ability to present antigens and can be used in certain diseases such as cancer. Contribute to the ability to fight.

  In further embodiments, RNA can be used for vaccine production. For example, dendritic cells may be transfected with RNA encoding a tumor antigen (Heiser et al., J. Clin. Invest. 109: 409-417, 2002; Mitchell and Nair, J. Clin. Invest. 106: 1065-1069, 2000). This approach overcomes the limitations of obtaining sufficient amounts of tumor material and extends treatment to patients who are otherwise excluded from clinical trials. For example, RNA molecules of the invention isolated from tumors can be amplified using RT-PCR. In certain embodiments, the RNA molecules of the invention are isolated directly from the tumor and transfected into dendritic cells without an intervening cloning step.

  In certain embodiments, the molecules of the invention are modified so that the peptide antigen is more antigenic than its native state. These embodiments address the need in the art to overcome the in vivo poor immunogenicity of most tumor antigens by enhancing the immunogenicity of tumor antigens through modification of the epitope sequence (Yu And Restifo, J. Clin. Invest. 110: 289-294, 2002).

  Another recognized problem with cancer vaccines is the presence of existing neutralizing antibodies. Some embodiments of the present invention overcome this problem by using viral vectors derived from non-mammalian natural hosts, such as fowlpox virus. Another embodiment that also bypasses existing neutralizing antibodies involves the use of genetically engineered influenza viruses and naked plasmid DNA vaccines that contain DNA that is not protein related (Yu and Restifo, J. Clin. Invest. 110: 289-294, 2002).

  All immunogenic methods of the invention can be used alone or in combination with other conventional or non-conventional treatments. For example, immunogenic molecules may be combined with other molecules having various antiproliferative effects, or with additional substances that aid in stimulating the immune response, ie adjuvants or cytokines.

  For example, in certain embodiments, the nucleic acid vaccine encodes an alphavirus replicase enzyme in addition to the tumor antigen. This approach to vaccine therapy has been successfully combined with therapeutic antigen production with induction of apoptotic death of tumor cells (Yu and Restifo, J. Clin. Invest. 110: 289-294, 2002).

  In certain embodiments, the molecules of the invention are involved in the control of cell growth, and the agents of the invention inhibit unwanted cell growth. Such factors are useful for treating disorders involving abnormal cell proliferation, including but not limited to cancer, psoriasis and scleroderma. Whether a particular factor and / or therapeutic regimen of the present invention is effective in reducing unwanted cell proliferation, eg, in the context of treating cancer, can be determined using standard methods. For example, the number of cancer cells in a biological sample (eg, blood, biopsy sample, etc.) can be determined. Tumor burden can be determined using standard radiological or biochemical methods.

  The molecules of the invention find use in immunotherapy of hyperproliferative disorders, including cancer, neoplasms and paraneoplastic disorders. That is, the molecules of the invention can correspond to tumor antigens of which more than 1770 have been identified to date (Yu and Restifo, J. Clin. Invest. 110: 289-294, 2002). Immunotherapy approaches include passive immunotherapy and vaccine therapy, and can achieve both genetic and antigen-specific cancer immunotherapy.

  Passive immunization approaches include antibodies of the invention against specific tumor associated antigens. Such antibodies can eradicate systemic tumors at multiple sites without destroying normal cells. In certain embodiments, the antibody is combined with a radioactive component as described above, thereby combining, for example, the ability of the antibody to specifically label a tumor with the lethal addition of a radioisotope to tumor DNA.

  Useful antibodies include discrete epitopes or combinations of nested epitopes, ie 10-mer epitopes, and all potential 8-mers and 9-mers, or related peptide multimers that incorporate overlapping epitopes ( Dutoit et al., J. Clin. Invest. 110: 1813-1822, 2002). Thus, a single antibody can interact with one or more epitopes. Furthermore, the antibodies may be used alone or in combination with various antibodies that all recognize either a single epitope or multiple epitopes.

  Neutralizing antibodies can provide treatment for cancer and proliferative disorders. A neutralizing antibody that specifically recognizes the protein or peptide of the present invention can bind to the protein or peptide, for example, in a body fluid or in the extracellular space, thereby reducing the biological activity of the protein or peptide. Adjust. For example, neutralizing antibodies specific for proteins or peptides that play a role in stimulating the growth of cancer cells may be useful for modulating the growth of cancer cells. Similarly, neutralizing antibodies specific for proteins or peptides that play a role in cancer cell differentiation may be useful in regulating cancer cell differentiation.

(MGD-CSF “knock-out” and homologous recombination)
Endogenous gene expression can be reduced by inactivating or “knocking out” the gene of interest and / or its promoter using targeted homologous recombination. For example, Smithies et al., Nature, 317: 230-234 (1985); Thomas et al., Cell, 51: 503-512 (1987); and Thompson et al., Cell, 5: 313-321 (1989). For example, a variant of the invention, a non-functional polynucleotide (or a DNA sequence that is not completely related) adjacent to DNA homologous to an endogenous polynucleotide sequence (either the coding region or the regulatory region of a gene) May be used with or without a selectable marker and / or a negative selectable marker to transfect cells to express a polypeptide of the invention in vivo. In another embodiment, techniques known in the art are used to generate knockouts in cells that contain, but do not express, the gene of interest. Insertion of the DNA construct via targeted homologous recombination results in inactivation of the target gene. Such an approach is particularly suitable for research and agriculture, where modifications to embryonic stem cells can be used to generate animal offspring with inactive target genes. For example, Thomas et al., Cell 51: 503-512 (1987); Thompson (1989), supra). However, this approach is routinely used for use in humans, provided that the recombinant DNA construct is administered or targeted directly to the required site in vivo using an appropriate viral vector that will be apparent to those skilled in the art. Can be adapted.

  In a further embodiment of the invention, cells that have been genetically engineered to express a polypeptide of the invention, or genetically engineered to express no polypeptide of the invention, such as a knockout, are obtained in vivo. Administer to patients. Such cells may be obtained from patients, including human and non-human animals, or MHC compatible donors and include, but are not limited to, fibroblasts, bone marrow cells, blood cells (eg, lymphocytes). , Fat cells, muscle cells, endothelial cells and the like. The cell is genetically engineered in vitro using recombinant DNA technology to introduce the coding sequence of the polypeptide of the invention into the cell, or without limitation, a plasmid, cosmid, YAC, naked DNA, electro Coding sequences associated with the polypeptides of the invention and / or by means of transduction (using viral vectors and / or vectors that incorporate transgenes into the cell genome) or transfection procedures, including the use of porations, liposomes, etc. Or destroy the endogenous regulatory sequences (control sequences). The coding sequence of the polypeptide of the invention may be under the control of a strong constitutive or inducible promoter or promoter / enhancer to achieve expression and secretion of the polypeptide of the invention. Engineered cells that express and secrete polypeptides of the invention can be introduced into a patient systemically, eg, into the circulation or into the peritoneal cavity. Alternatively, the cells can be incorporated into a matrix and transplanted into the body, for example, genetically engineered fibroblasts can be transplanted as part of a skin graft; genetically engineered endothelial cells are Can be transplanted as part of a lymphatic or vascular graft (see, eg, Anderson et al., US Pat. No. 5,399,349; and Mulligan & Wilson, US Pat. No. 5,460,959).

  If the cells to be administered are non-self or non-MHC compatible cells, they can be administered using well-known techniques that prevent the development of a host immune response against the introduced cells. For example, the cells may be introduced in an encapsulated form that allows for immediate exchange of components with the extracellular environment but does not allow the introduced cells to be recognized by the host immune system.

(Transgenic non-human animals)
The polypeptides of the present invention can also be expressed in transgenic non-human animals. Any species of animal including, but not limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows, and non-human primates such as baboons, monkeys and chimpanzees May be used to generate transgenic animals. In certain embodiments, the polypeptides of the invention are expressed in humans using techniques described herein or otherwise known in the art as part of a gene therapy protocol.

  Any technique known in the art can be used to introduce a transgene (represented in a polynucleotide shown in the sequence listing) into an animal to generate the founder strain of the transgenic animal. Such techniques include, but are not limited to, pronuclear microinjection (Patterson et al., Appl. Microbiol. Biotechnol. 40: 691-698 (1994); Carver et al., Biotechnology (NY) 11: 1263-1270 (1993). Wright et al., Biotechnology (NY) 9: 830-834 (1991); and Hoppe et al., US Pat. No. 4,873,191 (1989)); Retrovirus-mediated gene transfer into the germline (Van der Putten). Proc. Natl. Acad. Sci. 82: 6148-6152 (1985)), blastocysts or embryos; gene targeting in embryonic stem cells (Thompson et al., Cell 56: 313-321 ( 989)); electroporation of cells or embryos (Lo, Mol. Cell. Biol. 3: 1803-1814 (1983)); introduction of polynucleotides of the invention using a gene gun (eg, Ulmer et al., Science 259: 1745 (1993)); introduction of nucleic acid constructs into embryonic pluripotent stem cells and transfer of stem cells back to blastocysts; and sperm-mediated gene transfer (Lavitrano et al., Cell 57: 717-723 ( For a review of such techniques, see Gordon, Intl. Rev. Cytol. 115: 171-229 (1989), and US Pat. Capecchi et al., Positive-Negative Selecti n Methods and Vectors); U.S. Pat. No. 5,631,153 (Capecchi et al., Cells and Non-Human Organics Containing Predicted Genomic Modifications and PositiveMetics, United States No. 5,631,153) See also (Leder et al., Transgenic Non-Human Animals) and US Pat. No. 4,873,191 (Wagner et al., Genetic Transformation of Zygotes). Transgenic clones in which any technique known in the art, e.g., nuclear transfer from cultured embryos, fetuses or adult cells induced to a quiescent state into a nuclear-depleted oocyte, comprises a polynucleotide of the invention (Campbell et al., Nature 380: 64-66 (1996); Wilmut et al., Nature 385: 810-813 (1997)).

  The present invention provides transgenic animals that carry the transgene in their entire cells, as well as animals that carry the transgene to some extent but not its whole cells, such as mosaic animals or chimeric animals. This transgene can be incorporated as a single transgene, as in concatamers, or as multiple copies, eg, head-to-head tandem or head-to-tail (head-to-tail). -Tail) Tandem. This transgene can also be selectively introduced and activated into specific cell types, eg, according to the teaching of Lasko et al. (Proc. Natl. Acad. Sci. 89: 6232-6236 (1992)). The regulatory sequences (control sequences) required for such cell type specific activation depend on the particular cell type of interest and will be apparent to those skilled in the art. It may be desirable to incorporate the polynucleotide transgene into the chromosomal site of the endogenous gene, and then the gene target is appropriate. In short, when such a technique is utilized, a vector comprising several nucleotide sequences that are homologous to an endogenous gene can be integrated into the nucleotide sequence of the endogenous gene via homologous recombination with the chromosomal sequence, and Designed for functional purposes. A transgene can also be selectively introduced into a particular cell type, thereby inactivating the endogenous gene only in that cell type, eg, according to the teachings of Gu et al. (Science 265: 103-106 (1994). )). The regulatory sequences (control sequences) required for such cell-specific inactivation will be apparent to those skilled in the art depending on the particular cell type of interest.

  Once transgenic animals are generated, recombinant gene expression can be assayed using standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques, and animal tissue may be analyzed to confirm that transgene integration has occurred. The level of transgene mRNA expression in tissues of transgenic animals also includes, but is not limited to, Northern blot analysis, in situ hybridization analysis, and reverse transcription PCR (RT-PCR) of tissue samples obtained from the animals. It may be evaluated using techniques. Transgenic gene-expressing tissue samples can also be assessed immunocytochemically or immunohistochemically using antibodies specific for the transgene product.

  Once the founder animals are generated, they may be bred, inbred, outbred or crossed to produce a particular animal colony. Examples of such breeding strategies include, but are not limited to, cross-breeding of founder animals with two or more integration sites to establish another lineage; effect of additive expression of each transgene Thanks to this, inbreds of different strains to produce complex transgenic animals that express transgenes at high levels; predetermined integration to increase expression and eliminate the need for screening animals by DNA analysis Crossbreeding of homozygous transgenic animals to generate animals homozygous for the site; crossbreeding of another homozygous strain to generate complex heterozygous or homozygous lines; and purposes Breeding to place this transgene on a separate background that is appropriate for this experimental model.

  The transgenic and “knock-out” animals of the invention include, but are not limited to, biological functions of the molecules of the invention to study conditions and / or disorders associated with abnormal expression of the molecules of the invention. Have useful uses, including animal model systems, in making and screening for compounds effective in ameliorating such conditions and / or disorders.

(kit)
The present invention provides kits that can be used in the above methods. In one embodiment, the kit comprises an antibody of the invention, eg, a purified antibody, in one or more containers. In certain embodiments, the kits of the invention comprise a substantially isolated polypeptide comprising an epitope that is specifically immunoreactive with the antibodies contained in the kit. The kit of the present invention may also contain a control antibody that does not react with the polypeptide of interest.

  In certain embodiments, the kits of the invention include a means for detecting antibody binding to the polypeptide of interest. For example, the antibody may be conjugated to a detectable substrate, such as a fluorescent compound, enzyme substrate, reactive compound or luminescent compound, or a second antibody that recognizes the first antibody is detected. It may be conjugated to a possible substrate.

  In certain embodiments, the kit is a diagnostic kit for use in screening sera containing antibodies specific for MGD-CSF related molecules. Such a kit may comprise a control antibody that does not react with the polypeptide of interest. Such a kit can comprise a substantially isolated polypeptide antigen comprising an epitope that is specifically immunoreactive with at least one anti-polypeptide antigen antibody. Furthermore, such a kit comprises means for detecting the binding of this antibody to an antigen. The antibody may be conjugated to a fluorescent compound such as fluorescein or rhodamine that can be detected by flow cytometry. In certain embodiments, the kit may comprise a recombinantly produced polypeptide antigen or a chemically synthesized polypeptide antigen. The polypeptide antigens of the kit may also be bound to a solid support.

  In a further embodiment, the detection means of the above kit comprises a solid support to which the polypeptide antigen is bound. Such a kit may also comprise an unbound reporter labeled anti-human antibody. In this embodiment, antibody binding to the polypeptide antigen can be detected by binding of the reporter-labeled antibody.

  In a further embodiment, the invention includes a diagnostic kit for use in screening sera containing an antigen of a polypeptide of the invention. The diagnostic kit comprises a substantially isolated antibody that is specifically immunoreactive with a polypeptide or polynucleotide antigen, and means for detecting binding of the polynucleotide or polypeptide antigen to the antibody. In certain embodiments, the antibody is bound to a solid support. In certain embodiments, the antibody is a monoclonal antibody. The detection means of this kit may comprise a second labeled monoclonal antibody. Alternatively or additionally, the detection means may include a labeled, competing antigen.

  In one diagnostic configuration, test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the method of the present invention. After binding the specific antigen antibody to the reagent and removing unbound serum components by washing, the reagent is reacted with a reporter labeled anti-human antibody to the amount of anti-antigen antibody bound on the solid support. Accordingly, a reporter is bound to this reagent. The reagent is washed again to remove unbound labeled antibody and the amount of reporter associated with the reagent is determined. Typically, a reporter is an enzyme that is detected by incubating this solid phase in the presence of a suitable fluorometric, luminescent or colorimetric substrate.

  This solid surface reagent can be prepared by known techniques for attaching protein material to solid support materials such as polymeric beads, dip sticks, 96-well plates and / or filtration materials. These attachment methods generally include non-specific adsorption of proteins to the support or chemically active groups on the solid support, such as activated carboxyl groups, hydroxyl groups, or aldehyde groups. , And typically a covalent bond of a protein via a free amine group. Alternatively, streptavidin coated plates may be used with biotinylated antigen.

  Additional objects and advantages of the invention will be set forth in part in the following detailed description, and in part will be obvious from the detailed description, or may be learned by practice of the invention. The objects and advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. Further, advantages described in the text of the detailed description are not limited to the claimed invention itself, even if they are not included in the claims.

  It is to be understood that both the foregoing general description and the following detailed description are representative of the claimed invention, are exemplary and not limiting. Furthermore, it should be understood that the invention is not limited to the specific embodiments described, but can, of course, vary. Moreover, the terminology used to describe a particular embodiment should not be construed as limiting. This is because the scope of the present invention is limited only by the scope of the claims. The claims do not encompass embodiments in the expiring state.

  With respect to value ranges, the present invention encompasses each value between the upper and lower limits of this range to at least 10 times the lower limit unit, unless the context clearly indicates otherwise. Furthermore, the present invention encompasses any other mentioned intervening values. In addition, the invention also encompasses ranges excluding either or both of the upper and lower limits of this range, unless specifically excluded from the stated ranges.

  Unless defined otherwise, the meaning of all technical and scientific terms used herein are those commonly understood by one of ordinary skill in the art to which this invention belongs. Those skilled in the art will also recognize that any methods and materials similar or equivalent to the methods and materials described herein can also be used to practice or test the present invention. Moreover, all publications mentioned in this specification are incorporated by reference in their entirety.

  As used herein and in the appended claims, the singular forms “a”, “or”, and “this” refer to the context clearly. It should be noted that it includes multiple objects unless otherwise indicated. Thus, for example, reference to “a polypeptide of the present invention” includes a plurality of such polypeptides, and reference to “the agent” is known to those skilled in the art. Includes references such as one or more factors and equivalents thereof.

  Further, all numbers representing amounts, such as components, reaction conditions,% purity, polypeptide and polynucleotide lengths, as used herein and in the claims, are expressed as “about” unless otherwise indicated. ) ". Accordingly, the numerical parameters set forth in the specification and claims are approximations that may vary depending on the desired properties of the present invention. At a minimum, we do not intend to limit the application of the equivalent principle to the claims, but each numerical parameter applies normal rounding techniques to the reported number of significant digits. Should be interpreted at least. Nevertheless, the numerical values shown in the specific examples are reported as accurately as possible. Any numerical value, however, inherently contains certain errors from the standard deviation of its experimental measurement.

  The specification is most thoroughly understood in light of the references cited herein. Each of these references is hereby incorporated by reference in its entirety.

  The examples are intended to be exemplary of the present invention and therefore should not be construed as limiting the invention in any way, and aspects and embodiments of the invention discussed above. Details are provided and provided. This example does not indicate that the following experiments are all performed or the only experiments. Efforts have been made to ensure accuracy with respect to numbers used (eg amounts, temperature, etc.) but some experimental errors and deviations should be expected. Unless specified otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

(Example 1: Amino acid sequence alignment of MGD-CSF and MCG34647)
MGD-CSF and NP_689669 have T-COFFEE Version 1.37, cpu = 0.00 sec. , Score = 72, Nseq = 3, len = 242 and compared them by aligning their amino acids using the crustal form of the program. As shown in FIG. 1, the amino acid sequence of MGD-CSF is different from the amino acid sequence of NP_686969 (MCG34647). The latter sequence has a glutamine (Q) residue at amino acid 81 position. The five adjacent amino acid residues adjacent to and on either side of the NCBI sequence of MCG34647 are NVTRLQRAQVS (SEQ ID NO: 279). In contrast to this published sequence of MCG34647, MGD-CSF contains the amino acid sequence NVTRLRAQVS (SEQ ID NO: 280). Differences between these sequences arise from alternative splicing of the MCG34647 gene between exon 3 and exon 4. The genomic sequences at the boundaries of exons 3-4 are the codons aac gtc acc agg ctg gtg (SEQ ID NO: 281) and cag cag agg gcc cag gtg agc (SEQ ID NO: 282), where the gtg codon (shown in italics) is It corresponds to a 5 ′ splice donor at the end of exon 3, and the two cag codons (shown in italics) correspond to two alternative splice acceptor sites at the start of exon 4. Thus, the published MCG34647 sequence corresponds to the transcript resulting from the use of the first cag splice acceptor site, while the MGD-CSF sequence corresponds to the transcript resulting from the use of the second cag splice acceptor site. To do.

  The MCG34647 glutamine 81 residue is encoded by a second cag codon that is not spliced out when the first cag splice acceptor site is used. In contrast, the use of a second cag as a splice acceptor site results in splicing out the first cag sequence of the resulting RNA transcript, which in turn results in the corresponding glutamine in the MGD-CSF splice variant. Resulting in a deletion. MGD-CSF is therefore a splicing variant and represents an RNA and protein species that is distinct from MCG34647.

(Example 2: Plasmid vector for MGD-CSF expression)
The MGD-CSF gene was cloned into pTT-5 and pTT-2 mammalian expression vectors modified as shown in FIGS. 2 and 3 using standard cloning procedures. The MGD-CSF gene was also cloned into the pIB / V5His-DEST insect cell expression vector (Invitrogen, Carlsbad CA) using standard cloning procedures. The resulting constructs are listed in Tables 1 and 5. These include human MGD-CSF untagged in vector pTT5 (MGD-CSF), human MGD-CSF untagged in vector pTT2 (CLN00839395), human MGD- having a C-terminal V5H8 tag in vector pTT5. CSF (CLN00732663), human MGD-CSF tagged with V5H8 (CLN00732663), human MGD-CSF with C-terminal V5H8 tag in vector pTT2 (CLN00840351), human MGD with C-terminal V5H8 in vector pIB / V5His-DEST -CSF (CLN00758593), and human MGD-CSF with collagen secretion leader and C-terminal Streptag in vector pTT5-G CLN00816424), and the like.

  To monitor MGD-CSF expression and secretion and to assist in its purification, construct CLN00821867 was generated at the Tobacco Etch Virus (TEV) protease recognition site engineered between the protein and the C-terminal truncation tag. . The seven amino acid recognition sites of TEV protease are Glu-Asn-Leu-Tyr-Phe-Gln-Gly (SEQ ID NO: 183) and cleavage occurs between Gln and Gly. The construct CLN00821867 was named MGD-CSF (1-241aa) _TEV_V5_StrepttagII_H8 and C-tagged in the vector pTT5-I.

  In order to improve the secretion of MGD-CSF, its secretory signal peptide encoded by the first 20 amino acids was replaced by 23 amino acids encoding the collagen signal peptide (GenBank protein accession number NP_001842). This construct, CLN00848149, is named MGD-CSF collagen SP (1-23aa) _MGD-CSF (21-241aa) and is untagged in the vector pTT5-G. Another such construct also has a TEV protease recognition site engineered between the protein and the C-terminal truncation tag. CLN00816424 is named MGD-CSF collagen SP (1-23aa) _MGD-CSF (21-241aa) _TEV_V5_StrepttagII_H8 and C-tagged in the vector pTT5-G. A third such construct was generated with a TEV site engineered between the N-terminal tag and the protein. CLN00816425 is named MGD-CSF collagen SP (1-23aa) _H8_StrepttagII_V5_TEV_MGD-CSF (21-241aa) and is N-tagged in the vector pTT5-H.

  Deletion constructs were generated in which amino acids were deleted from the N-terminus, C-terminus, or both ends of the mature protein. The MGD-CSF signal peptide of these deletion constructs was replaced with a collagen signal peptide. CLN00848160 lacks 25 N-terminal amino acids; it is untagged in the vector pTT5-G, termed MGD-CSF collagen SP (1-23aa) _MGD-CSF (26-241aa) . CLN00848173 lacks 30 N-terminal amino acids; it is untagged in the vector pTT5-G, termed MGD-CSF collagen SP (1-23aa) _MGD-CSF (31-241aa) . CLN00848209 lacks 5 C-terminal amino acids and 20 N-terminal amino acids (signal peptide); it is named MGD-CSF collagen SP (1-23aa) _MGD-CSF (21-236aa) And untagged in the vector pTT5. CLN00848197 lacks 10 C-terminal amino acids and 20 N-terminal amino acids (signal peptide); this is named MGD-CSF collagen SP (1-23aa) _MGD-CSF (23-231aa) And untagged in the vector pTT5. CLN00848185 lacks 28 C-terminal amino acids and 20 N-terminal amino acids (signal peptide); this is named MGD-CSF collagen SP (1-23aa) _MGD-CSF (21-213aa) And untagged in the vector pTT5. CLN00848220 lacks 25 N-terminal amino acids and 10 C-terminal amino acids; it is named MGD-CSF collagen SP (1-23aa) _MGD-CSF (26-231aa) and is designated as vector pTT5 Untag in.

  Two mouse orthologs of MGD-CSF were identified and pTT5-I (tagged with a TEV site between untagged pTT5 (CLN00840257 and CLN00847948) and between this clone and tag by standard procedures. CLN00840253 and CLN00842712). These orthologs may be used to perform animal studies on the biological activity of MGD-CSF in mouse tissues and cells.

  The mouse ortholog represented by constructs CLN00840257 and CLN00840235 was derived from Mus musculus adult male small intestine cDNA clone 2010004A03 from the RIKEN full-length enriched library; theoretical protein 12842043; locus AK008082. Construct CLN00840257 corresponds to the open reading frame (ORF) of the nucleotide sequence of phantom clone 2010004A03 and is a mouse ortholog of human MGD-CSF cloned into vector pTT5. The construct CLN00840253 corresponds to the ORF of the nucleotide sequence of phantom clone 201404A03 and was cloned into the vector pTT5-I.

  The mouse ortholog presented by the constructs CLN00847948 and CLN00842712 was derived from the Musken mouse clone 2010004A03 from the RIKEN full-length enriched library, mRNA (cDNA clone MGC: 28891 IMAGE: 4912097), complete cds18921436 at the locus BC016254. The construct CLN00847948 (18921436) corresponds to the ORF nucleotide sequence of human MGD-CSF cloned into the vector pTT5. This construct CLN00842712 (18921436) corresponds to the ORF nucleotide sequence of human MGD-CSF cloned into the vector pTT5.

(Example 3: Transient expression in mammalian cells)
Complementary DNA encoding MGD-CSF polypeptide was cloned into expression vectors pTT5 and pCDNA-pDEST40 and expressed as both tagged and untagged proteins. Protein levels were quantified by measuring the level of tags, such as V5His tag, by quantitative Western blot analysis. The expression vector was used to generate adherent 293 cells using the transfection agent Fugene 6 (Roche, Nutley NJ) contained in DMEM containing 10% fetal bovine serum (FBS) and penicillin / streptomycin (100 μg / ml, 100 U / ml). Transfected in and incubated at 37 ° C. in 5% CO ₂ for 40 hours, after which the cells were washed with PBS and incubated for a further 48 hours in complete DMEM. Cell supernatants were collected, cell debris removed by centrifugation, and tested for biological activity (untagged cDNA) and protein expression (V5-tagged cDNA) by Western blot assay using anti-V5 antibody. .

Expression studies were also performed on 293-6E cells transiently expressing the tagged MGD-CSF construct in suspension culture. Cells were diluted to a density of 6 × 10 ⁵ cells per ml in 25 ml FreeStyle medium (Invitrogen, Carlsbad CA) 18-24 hours prior to transfection. Transfection complexes are made by adding 25 μg of DNA to 1.25 ml of PBS, adding 50 μl of linear 25 kD polyethyleneimine (PEI) (Polysciences, Warrington PA) dissolved in water at a concentration of 1 mg / ml. And mixing the solution and incubating the mixture for 1 hour at room temperature before adding it to the cells to be transfected. Cells and their supernatants were harvested 3-6 days after transfection and protein expression was assessed by Western blot analysis.

Cell suspension (1 ml) was pelleted and then mixed with 4 parts XT sample buffer (Bio-Rad, Hercules CA). After denaturation at 99 ° C. for 3 minutes, samples were loaded onto Criterion XT SDS-PAGE gels (Bio-Rad, Hercules CA) or stored at −20 ° C. Cell pellets were treated with 100 μl lysis buffer (1% NP-40; 50 mM Tris-HCl, pH 8.0; 150 mM NaCl; and 1 tablet complete protease inhibitor (Roche, Indianapolis).
IN)) and dissolved by resuspension. Lysed cells were pelleted by centrifugation at 14,000 rpm, and the resulting clear lysate protein and cell suspension were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Western blots using HRP-conjugated monoclonal antibodies specific for the V5 epitope (Invitrogen, Carlsbad CA), or polyclonal rabbit anti-MGD-CSF antibody (Five Prime Therapeutics, Inc., South San Francisco CA). This was done by probing the membrane. Bound rabbit anti-MGD-CSF antibody was detected with a polyclonal goat anti-rabbit conjugated to horseradish peroxidase (Jackson Immuno Research, West Grove PA). Immune complexes were visualized by incubating the membrane in a chemiluminescent substrate (SuperSignal West Femto, Pierce, Rockford IL) and exposing it to a photosensitive film.

  As shown in FIGS. 4A and 4B, 293-6E cells expressed tagged MFD-CSF 3-6 days after transfection. FIG. 4A shows the expression of MGD-CSF tagged with V5H8 (CLN00732663) transiently transfected into 293-6E cells. This left panel shows intracellular MGD-CSF. Expression was almost prominent 3 days after transfection. The middle panel shows MGD-CSF secreted into the supernatant. Expression was most prominent 6 days after transfection. The right panel shows a quantitative positive control (Positope, Invitrogen, Carlsbad, CA).

  FIG. 4B shows the expression of MGD-CSF with a collagen secretion sequence and tagged with V5H8 (CLN00816424). The left panel shows intracellular MGD-CSF. Expression was observed at 3 days and continued to increase over 6 days after transfection. The middle panel shows MGD-CSF secreted into the supernatant; its expression also increased from 3 to 6 days. The right panel shows a quantitative positive control (Positope, Invitrogen, Carlsbad, CA).

  In both FIGS. 4A and 4B, the protein load of the cells and supernatant was adapted to reflect the number of cells comparable to the gel load in the left and middle panels. Thus, the amount of MGD-CSF shown in the middle panel reflects the secretory efficiency of the cells. The targeted protein detected in the supernatant had a molecular weight of approximately 40 kD, while the intracellular protein had a molecular weight of approximately 37 kD, probably due to incomplete glycosylation. The yield of secreted protein varied depending on the design of the construct. CLN00732663 was expressed in cells at low yield. Although it was 37 kD and not 40 kD, it was still detected in the cell culture supernatant 6 days after transfection, probably due to cell lysis, also in low yield (about 5-10 ng / ml). Replacing the endogenous signal peptide with an exogenous secretion signal and an extended cleavable C-terminal tag increased expression and secretion at least 1-fold (CLN00816424). In addition, only higher molecular weight protein bands can be detected in the supernatant of cell cultures transfected with CLN00816424, indicating that this protein was secreted and lysed cells It was not derived.

Example 4: Growth and viability of transfected cells
As shown in FIG. 5A and Table 1, 293-6E cells transiently transfected with CLN00816424 continued to grow for 3-6 days after transfection. The density of cells in suspension cultures was determined using tryptic plates 3 to 6 days after transfection with CLN00542945 (black), CLN00732663 (light gray), CLN00821867 (hatched), and CLN00816424 (grid). Cells that were excluded of blue were monitored by counting and compared to a control gene (dark gray) encoding secreted alkaline phosphatase (SEAP). Both control SEAP cells and cells transfected with CLN00816424 (MGD-CSF with collagen leader) increased in number over 3-6 days. Cells transfected with CLN00542945 (untagged MGD-CSF), CLN00732663 (V5H8 tagged MGD-CSF), or CLN00821867 (strep tagged MGD-CSF) did not grow.

  As shown in FIG. 5B, cells transiently transfected with CLN00816424 (MGD-CSF with collagen leader) remained viable. They maintained viability in excess of 80% in culture for 6 days. Cell viability in suspension cultures was measured using a hemocytometer over 3-6 days after transfection with CLN00542945 (black), CLN00732663 (light gray), CLN00821867 (hatched), and CLN00816424 (grid). Monitored by trypan blue exclusion and compared to a control gene (dark gray) encoding secreted alkaline phosphatase (SEAP). Cells expressing MGD-CSF, CLN00732663 and CLN00821867 showed increased cytotoxicity as evidenced by a decrease in cell viability over time in culture. This toxicity is not specific for MGD-CSF cDNA in host 293 cells, but is only observed under certain culture conditions.

(Example 5: Stable transfection with mammalian cells)
MGD-CSF was stably expressed by transfected adherent 293-T cells. Stable transfections were performed on 293-T cells purchased from ATCC (Manassas VA), complete DMEM medium (10% FBS (Mediatech, Herndon VA); 100 U / ml penicillin, 100 μg / ml streptomycin and 2 mM Cultured in glutamine (DMEM medium supplemented with Invitrogen, Carlsbad CA). The day before transfection, 1.25 × 10 ⁵ cells were seeded in T-175 culture flasks (Corning, Acton MA) and incubated overnight at 37 ° C. with 5% CO ₂ . Cells were transfected by mixing 114 μl Fugene 6 (Roche, Nutley NJ) and 1.9 ml RPMI-1640 medium (Mediatech, Herndon VA) and incubated at room temperature for 5 minutes. Plasmid DNA (19 μg full length MGD-CSF in pIRESpuro3) (BD Biosciences, San Jose, Calif.) Was added to the Fugene / medium mixture and incubated for 15 minutes at room temperature. The lipid / DNA mixture was transferred to a T-175 flask and incubated with the cells for 16 hours. The next day, cells were detached with 0.25% trypsin (Invitrogen, Carlsbad CA) and grown in three T-175 flasks. After about 16 hours, the cells were allowed to bind to the culture vessel and the selection reagent puromycin (Invitrogen, San)
(Diego CA) was added to a final concentration of 10 μg / ml. The selection medium was changed once a week and cell viability was monitored for 4-6 weeks. Expression was confirmed by Western blot analysis using the polyclonal rabbit anti-MGD-CSF antibody described above.

Adherent 293-T cells stably expressing MGD-CSF were adapted for suspension culture in low serum or serum-free medium. Cells were resuspended in FreeStyle medium (Invitrogen, Carlsbad CA) or Hyq PF CHO LS medium (Hyclone, Logan UT) at a concentration of 10 ⁶ / ml, respectively, and 5% FBS (Mediatech, Herndon, VA). ). Suspension cell cultures were maintained at a volume of 50 ml in 250 ml shake flasks and cultured at 37 ° C. and 5% CO ₂ . This medium was changed twice a week to maintain the cells at a density of about 10 ⁶ / ml or less. Cell viability was measured by trypan blue exclusion. When viability exceeded 80%, the serum concentration was gradually reduced to 3%, 2%, 1% and then no serum. Transfected cells were adapted to serum-reduced or serum-free conditions. The cell viability and protein yield of 293-T cells transiently expressing untagged MGD-CSF were 3% FBS after 4 days in culture in HyQ-CHO medium with 1% FBS. It was significantly higher than the viability and yield observed after 6 days of transient expression in 293-E cells cultured in Free Style medium with

  Adherent 293-T cells maintained in FreeStyle medium with 3% FBS proliferated relatively slowly; their viability was 67% after 3 passages. In comparison, cells maintained in HyQ PF CHO LS medium with 1% FBS proliferated more rapidly; their viability exceeded 85% after 4 passages. As shown in FIG. 6A, protein yield (right panel) of cultures in HyQ PF CHO LS medium and 1% FBS as determined by quantitative Western blot, FreeStyle medium supplemented with 3% FBS. Increased more than 10 times compared to the yield from cells cultured in (left panel).

  As shown in FIG. 6B, stably transfected MGD-CSF 293-T adapted to grow in suspension culture in low or no serum medium as shown by Western blot analysis. High protein yields were obtained from the cells. Stablely transfected MGD-CSF 293-T cells adapted to grow in suspension culture with low serum secreted more than 8 times (1.3 μg ml) (panel 1). Cells grown in serum-free medium are more stable than stably transfected MGD-CSF 293-T cells (approximately 160 ng / ml) that adhere and proliferate (panel 3) as described above over 4 days (panel 3). Within 6 days, more than 4-fold (650 ng / ml) (panel 2) MGD-CSF was secreted into the culture supernatant. MGD-CSF is an E.I. It was expressed in E. coli and then purified to serve as a quantitative standard (panel 4).

(Example 6: Bioreactor fermentation)
As shown in FIG. 7, bioreactor fermentation improved MGD-CSF productivity to about 10 μg / ml during the course of 6-7 days fermentation in a 10 liter bioreactor. Cells adapted to grow in low serum were seeded at a concentration of about 5 × 10 ⁵ / ml in 10 liters of HyQ PF CHO LS medium supplemented with 1% FBS. The fermentation was monitored for 6 days and samples were prepared for gel electrophoresis as described above. The left panel of FIG. 7 shows the presence of MGD-CSF in gels loaded with 22.5 μl per lane and stained 1-6 days after inoculation. Bovine serum albumin (BSA) is shown as a quantitative control in the right panel. The arrow indicates the position of MGD-CSF.

(Example 7: Protein isolation)
MGD-CSF was isolated from 293T cells grown in suspension culture stably expressing the protein as described above and as shown in FIG. The culture supernatant was adjusted to 0.5 M NaCl, pH 5.0 (with HCl), and then concentrated 5-fold using a cellulose membrane (Millipore, Billerica MA) with a molecular weight cut-off of 10 kD. The concentrate was dialyzed against buffer A (10 mM acetate pH 5.0, 110 mM NaCl) and fractionated on an SP-Sepharose FF (Amersham, Piscataway NJ) cation exchange column equilibrated with buffer A. did. The protein was eluted with a linear gradient to 1.5 M NaCl and the fraction containing MGD-CSF was dialyzed against buffer B (10 mM 1,3 diaminopropane pH 8.9, 30 mM NaCl).

This dialyzed SP-Pool is added to a Heparin Sepharose HP (Amersham, Piscataway, NJ) column equilibrated with Buffer B, and 1.5M
Fractions containing MGD-CSF were dialyzed against buffer C (10 mM bis-tripropane pH 7.4, 30 mM NaCl), eluting with a linear gradient to NaCl. The dialyzed Hep-Pool was fractionated on a Q-Sepharose FF (Amersham, Piscataway, NJ) anion exchange column equilibrated in buffer C and eluted with a linear gradient to 1.5M NaCl. Fractions containing MGD-CSF were pooled and the Q-Pool was snap frozen in liquid nitrogen and stored at -80 ° C. This purification procedure recovered a 12% yield of the expressed protein with a purity greater than 95%.

(Example 8: Mutation analysis of cysteine to serine)
The MGD-CSF sequence contains seven cysteine residues located at amino acid positions 35, 167, 176, 178, 179, 190 and 198. Based on a comparison of denaturing and non-denaturing gel electrophoresis results under non-reducing conditions, MGD-CSF does not form disulfide-linked oligomers. Thus, at least one of the cysteine residues in the native protein is expected to be unpaired. Unpaired cysteine residues can lead to inappropriate protein folding and formation of covalent aggregates.

  A set of 7 muteins (muteins) of the MGD-CSF protein was constructed, expressed and characterized, where each of the cysteines was mutated to serine to understand its disulfide bond pattern. By analyzing this disulfide bond pattern, the elimination of one or more free cysteine residues results in improved properties such as improved expression and secretion from mammalian cells, reduced aggregation of purified proteins, and / Or E. It can be determined whether to produce MGD-CSF proteins that have the ability to produce active recombinant MGD-CSF when expressed in E. coli.

  DNA encoding each mutein (mutein) was generated from a construct containing a collagen signal peptide and a nucleotide sequence encoding mature MGD-CSF (CLN00848149). The protein was expressed in 293-T cells as described above. The supernatant was collected and subjected to reducing and non-reducing gel electrophoresis followed by Western blotting with a polyclonal antibody raised against the middle peptide epitope in human MGD-CSF.

  As shown in FIG. 8B, Western blots of reducing gels were analyzed to determine the relatives of wild-type human MGD-CSF (with native or collagen signal peptide) and each Cys to Ser mutein (mutein). The expression level was determined. It was observed that the secretory protein yield of wild type human MGD-CSF was higher with the collagen signal peptide than with the natural signal peptide. All muteins (muteins) were observed to have at least a slight decrease in the yield of secreted protein when compared to wild-type human MGD-CSF with a collagen signal peptide. The yields of C179S and C190S were significantly reduced and C35S expression was undetectable. Based on these results, C35, C179 and C190 are thought to be involved in disulfide bonds in native MGD-CSF.

  As further shown in FIG. 8B, Western blots of non-reducing gels were analyzed for changes in apparent molecular weight as determined by the relative migration of MGD-CSF species compared to protein standards. When the disulfide bond is broken, the apparent size of the protein typically increases under denaturing conditions of SDS-PAGE, and the magnitude of this increase is typically two in the primary sequence of the protein. Correlates with the distance between cysteine residues. Breakage of disulfide bonds can result in the formation of high molecular weight aggregated species. C167S is observed to have the same migration time as wild type MGD-CSF, but all other muteins (muteins) have altered migration behavior. This indicates that C167 is likely to be the only unpaired cysteine in native MGD-CSF. Both C179S and C190S initially form higher molecular weight species. These muteins (muteins) have the same change in protein yield and migration, suggesting that they are paired with each other in native MGD-CSF. C176S and C178S show the same slight decrease in migration, suggesting that they may be paired with each other in native MGD-CSF. Eventually, C198S has a greater change in migration, suggesting that its partner may be C35 located far away in the protein sequence. Due to the fact that C167 is thought to be the only unpaired cysteine in native MGD-CSF, it can be mutated to serine or alanine, improving the yield of the protein expressed here and of the recovered protein product A reduction in heterogeneity occurs.

(Example 9: MGD-CSF promotes hematopoietic cell proliferation)
(A. MGD-CSF promotes NK cell proliferation)
Mouse NK cells were purified from the spleens of C57BL6 10 week old female mice using the NK cell isolation kit according to the manufacturer's instructions (Miltenyi Biotechnology Inc., Auburn CA). Approximately 30,000 purified NK cells were incubated with purified MGD-CSF at a concentration of 0.01-10 μg / ml. The number of NK cells was determined using CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega # G7571). As shown in FIG. 9, after 4 days of incubation in RPMI with 5% FBS, MGD-CSF specifically increased NK cell number proliferation and / or survival in a dose-dependent manner.

Human NK cells were isolated and purified from blood enriched in capsulated cells obtained from Stanford Blood Center (Palo Alto, Calif.). The blood was diluted approximately 1: 5 with PBS and Ficoll (Ficoll-Paque Plus, Amersham Biosciences; Piscataway, NJ) was added to multiple 50 ml conical tubes each containing 25 ml of diluted blood (12. 5 ml / tube). The Ficoll / blood mixture was centrifuged at 450 × g for 30 minutes. The peripheral blood mononuclear cell (PBMC) layer was removed and washed with DPBS 1 × (Mediatech, Inc., Prince William Co. VA) without calcium and magnesium and pelleted at 1000 RPM for 10 minutes. PBMC were washed 3 times in PBS by centrifugation at 1350 RPM for 10 minutes to give 0.5% fetal calf serum (Gibco (Invitrogen), Carlsbad CA) and 2 mM EDTA (Sigma).
Aldrich, St. Resuspended in 40 ml PBS (Louis MO).

NK cells are human NK cell Isolation as recommended by the manufacturer.
Concentrated from PBMC containing Kit II (Miltenyi Biotechnology Inc., Auburn CA). This enrichment step utilized the “deplete” program of autoMACS ^™ Separator (Miltenyi Biotechnology Inc., Auburn CA); negative fractions corresponding to enriched NK cells were collected from the output port “neg1”. These cells were centrifuged at 1350 RPM for 10 minutes. The cell pellet was resuspended in DMEM containing 10% fetal calf serum and diluted to a concentration of 1 × 10 ⁶ cells / ml. The cells were placed in a 96 well round bottom plate at a concentration of 5 × 10 ⁴ cells in 50 μl DMEM containing 10% fetal calf serum per well at 37 ° C. in an atmosphere of 7% CO _2. Incubated with the controls and test factors described below for days.

The effects of control and test factors on the growth of human NK cells prepared in this manner are described in Promega CellTitreGlo Technical Bulletin.
No. 288 (Promega, Madison WI) was confirmed in a screening assay by measuring the number of viable cells in culture based on quantification of ATP by measuring luciferase activity. Quantitative results were read with an Lmax plate reader (Molecular Devices, Sunnyvale CA) at 0.6 seconds per well at room temperature. The ATP content of the wells was measured for 4 days after the plate.

  As shown in FIG. 10, conditioned media from cells transfected with MGD-CSF and cells transfected with plasmid DNA from cluster 190647, the source of MGD-CSF clones, produced NK cell generation. I was stimulated. Interferon γ (IFN-γ), interleukin 1 (IL-1) and GM-CSF were used as interferon positive controls. An external positive control containing recombinant interleukin 15 and an external negative control containing culture medium are shown on the right and left of FIG. This screening assay identified MGD-CSF as a factor that stimulates the generation of NK cells and / or stabilizes the number. This result was seen in 4 independent repeats of the screening assay. MGD-CSF does not consistently induce cytokine production from NK cells. This increased or stabilized the number of monocytes, but did not induce the production of cytokines from monocytes. MGD-CSF did not affect the number of activated T or B cells.

(B. MGD-CSF promotes hematopoietic stem cell proliferation)
MGD-CSF also stimulated the proliferation of bone marrow CD34 ⁺ hematopoietic stem cells (HSC cells) (Cambrex, Inc., Baltimore MD) in culture. As shown in FIG. 11, MGD-CSF increased proliferation in a dose-dependent manner. HSC cells were supplemented with 5% heat inactivated fetal calf serum (ATCC) and 10 ng / ml recombinant human stem cell factor (SCF), 10 ng / ml Flt3 ligand (Flt3L) (R & D Systems, Minneapolis MN), 1 ml / well. 1-2 in a 5% CO ₂ incubator at a density of 2.4 × 10 ⁴ cells per well in a 12-well tissue culture dish containing RPMI (ATCC) in a 5% CO ₂ incubator. Grows weekly in culture, washed with PBS, peeled off with 0.5 ml Versene (Gibco BRL, Gaithersburg MD), washed again with PBS, 1 ml PBS / 0.1% BSA (Sigma, St .Louis MO) and counted with a hemocytometer. Purified MGD-CSF increased its growth from 20 ng / ml to 500 ng / ml in a dose-dependent manner. MGD-CSF induced stem cell proliferation to a greater extent than M-CSF and to a similar extent to G-CSF and GM-CSF.

(C. MGD-CSF promotes myeloid cell proliferation in vitro)
Human primary monocytes were purified from PBMC using a protocol modified as previously described (de Almeida et al., Mem. Inst Oswaldo Cruz 95: 221-223, 2000). To isolate human PBMC from blood, the buffy coat was diluted in 6 volumes of PBS and then layered into 20 ml Ficoll in a 50 ml tube. The tube was centrifuged at 2,000 rpm for 20 minutes at 22 ° C. without the use of a centrifugal brake. PMBC cells were collected from the interface, washed twice with PBS, then resuspended in RPMI 5% FBS and filtered through a BD Falcon cell strainer. To purify primary unbound monocytes from PBMC, 6 ml of PBMC suspension (containing 70-120 × 10 ⁶ cells) was carefully and slowly layered onto 10 ml of hyperosmotic Percoll. The cells were centrifuged for 15 minutes at a speed of 580 × g without the use of a centrifuge brake. Interface cells were collected and washed with 50 ml RPMI 5% FBS. The purified monocyte cell pellet was resuspended in 50 ml RPMI 5% FBS.

  The monocyte assay was performed by incubating approximately 30,000 purified monocytes with MGD-CSF purified as described above. After 4 days of incubation in RPMI with 5% FBS, monocyte proliferation was determined using the CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega # G7571).

Conditioned medium from MGD-CSF transfected 293-T cells (MGD-CSF CM) promoted monocyte proliferation. The screening assay was performed in duplicate plates in a 96 well plate format on monocytes activated by mouse IgG2a. Table 6 shows a semi-quantitative description of the potency of activity that each clone stimulates monocyte proliferation and the degree of expression of each construct. MGD-CSF CM stimulated monocyte proliferation to approximately the same extent as GM-CSF. Results were considered significant if at least 2 standard deviations from the medium. The observed ED ₅₀ was 3-5 ng / ml. Mutant MGD-CSF protein was also tested in this assay. Mutant clone CLN00848185 demonstrates activity (titer) comparable to wild type protein, and other mutant clones have slightly lower activity than wild type protein, indicating that some mutant proteins It suggests that it can be used as a therapeutic protein.

  As shown in FIG. 12A, the stimulatory effect of MGD-CSF CM on monocyte proliferation was dose dependent over a 10,000-fold range. The lowest dose of MGD-CSF CM tested, 0.01 μl, had no significant effect on monocyte proliferation. A 10-fold increase in the dose of MGD-CSF CM to 0.1 μl induced cell proliferation to a significant level compared to the control. Further increase in dose to 1 μl MGD-CSF CM and 10 μl MGD-CSF CM further increased monocyte proliferation in a dose-dependent manner. No dose dependency was observed with either the empty vector or the negative controls CLN003732 or FPT026. The effect of a single dose of 10 ng / ml of GM-CSF, a stimulation positive control is shown, as well as the effect of negative control IL-10 and the effect of non-conditioned medium.

  As shown in FIG. 12B, both conditioned media from cells transfected with purified GM-CSF and MGD-CSF stimulated human monocyte proliferation. Thus, MGD-CSF functions as an agonist of monocyte proliferation in addition to its role in myeloid cell and granulocyte differentiation and proliferation. This can be used as a hematopoietic factor to enhance the recovery of hematopoietic cells after chemotherapy or radiation therapy and bone marrow transplantation in cancer patients.

(D. MGD-CSF promotes hematopoietic cell proliferation in vivo)
To understand the role of MGD-CSF in vivo, C57BL6 mice were treated with Wang et al., Cancer Res. 63: 9016-9022,2003 was injected with MGD-CSF plasmid DNA. Human cytochrome P450
The 3A4 promoter was operably linked to a nucleic acid molecule having the nucleotide sequence of MGD-CSF and injected into the tail vein of mice to transfect the mouse liver with MGD-CSF. The human 3A4 promoter was used to drive the expression of MGD-CSF in mouse liver. Complete blood count (CBC) and different analyzes were performed in control and experimental groups in each of two independent experiments using a Serono Baker 9000 hematology analyzer.

  The control group (Table 7A, animals 1-3) in the first experiment consisted of 3 uninjected mice age-matched to the experimental mice. The experimental group in the first experiment (Table 7A, animals 4-6) consisted of 3 mice injected with naked MGD-CSF DNA via the tail vein. Blood samples were collected 14 days after injection. As shown in Table 7A, injected mice had an elevated monocyte count compared to controls. Control animals 1, 2 and 3 had 94, 84 and 52 monocytes per μl of blood, respectively. Experimental animals 4 and 6 had 216 and 268 monocytes per μl of blood, respectively. Animal 3 did not yield a meaningful monocyte count.

  The control group in the second experiment (Table 7B, animals 1-6) consisted of 6 mice age-matched to the experimental mice and injected with the LacZ construct via the tail vein. The experimental group in the second experiment (Table 7B, animals 7-12) consisted of 6 mice injected with naked MGD-CSF DNA via the tail vein. Blood samples were collected 21 days after injection. As shown in Table 7B, injected mice had an elevated monocyte count compared to controls. None of the control animals had detectable monocyte levels. Four of the six experimental animals had detectable monocyte levels ranging from 50 to 78 monocytes per μl. These results demonstrate that MGD-CSF increased bone marrow cell number in vivo.

(Example 10: FACS analysis of the influence of MGD-CSF on hematopoietic differentiation)
In vitro granulocyte, monocyte and dendritic cell development assays further reveal the function of MGD-CSF in hematopoiesis. Human bone marrow CD34 ⁺ hematopoietic stem cells (HSC cells) (Cambrex, Inc., Baltimore MD) were cultured as described above.

Differentiation was confirmed by fluorescence activated cell sorter (FACS) analysis using fluorescently labeled antibodies to detect various markers on the granulocyte cell surface (Kavathas et al., Proc. Natl. Acad. Sci. 80: 524-528 (1983)). After 1 week of culture with or without MGD-CSF, G-CSF, GM-CSF or M-CSF, BM CD34 ⁺ cells were washed once with PBS and 0.5 ml of Versene (Gibco BRL, Gaithersburg MD), washed with 1 ml PBS / 0.1% BSA (Sigma, St. Louis MO) and resuspended in 0.2 ml PBS / 0.1% BSA (Sigma, St. Louis MO) Cloudy and aliquoted into 96 well plates for FACS staining (50 μl per well). The cells were incubated with fluorescent conjugated antibody for 15 minutes at 4 ° C. After two washes with 150 μl PBS / 0.1% BSA, cells were analyzed on a FACS Calibur according to the manufacturer's instructions (Bacton Dickinson, Franklin Lakes NJ). 10 ng / ml G-CSF, 10 ng / ml M-CSF or 30 ng / ml GM-CSF (from R & D Systems, Minneapolis MN) served as a positive control for the effects of known growth factors. Fluorescently labeled antibodies specific for specific surface markers of granulocytes, monocytes or dendritic cell lineages can be purchased from BD Biosciences (San Jose, Calif.) And introduced into the lineage of granulocytes, monocytes and dendritic cells. Used to confirm the effect of MGD-CSF on HSC cell differentiation.

(A. Granulocyte differentiation)
As shown in FIG. 13, MGD-CSF stimulated granulocyte differentiation from undifferentiated cells into differentiated granulocytes carrying different markers CD67 ⁺ and CD24 ⁺ . As a negative control, the baseline level of granulocyte differentiation in the presence of empty vector and absence of cytokines was determined to be 1.2%. A positive control, G-CSF, stimulated to differentiate 55% of granulocytes. MGD-CSF CM stimulated to differentiate 41% of granulocytes (arrow). The effect of MGD-CSF was synergistic with that of G-CSF, and in the presence of both, 64% of granulocytes were stimulated to differentiate.

CD24 and CD15 antibodies were used to monitor granulocyte differentiation. The CD24 antibody reacted with a 35-45 kDa 2-chain glycoprotein expressed on the surface of B cells and granulocytes. The CD15 antibody reacted with 3-fucosyl-N-acetyllactosamine (3-FAL), a 220 kDa carbohydrate structure also known as X-hapten. 3-FAL was expressed in 95% of the granulocytes tested including neutrophils and eosinophils and to varying degrees in monocytes but not in lymphocytes or basophils. CD15 plays a role in mediating phagocytosis, bactericidal activity and chemotaxis. Cells positive for both CD24 and CD15 correspond to granulocytes differentiated from BM CD34 ⁺ hematopoietic progenitor cells.

As shown in FIG. 14, 20 ng / ml and 100 ng / ml of MGD-CSF induces 3% differentiation to CD15 ⁺ / CD24 ⁺ granulocytes, and 500 ng / ml of MGD-CSF is CD15 ⁺ / CD24 ^+. 8% differentiation was induced into granulocytes. Both positive control G-CSF and GM-CSF induced 12% of bone marrow CD34 ⁺ cells to differentiate into CD15 / CD24 positive granulocytes.

(B. Monocyte differentiation)
As shown in FIG. 15, MGD-CSF stimulated monocyte differentiation from undifferentiated cells to differentiated monocytes carrying the CD14 ⁺ marker. As a negative control, the baseline level of monocyte differentiation in the presence of empty vector and in the absence of cytokines was determined to be 24%. GM-CSF stimulated to differentiate 20% of monocytes. MGD-CSF stimulated to differentiate 45% of monocytes (arrows). GM-CSF alone had no effect on these cells, but when combined with MGD-CSF, the effect was synergistic; in the presence of both, 55% of monocytes seem to differentiate. Stimulated by.

As shown in FIG. 16, monocyte differentiation was monitored using CD14 and CD16 antibodies. The CD14 antibody reacted with a 53-55 kD glycosylphosphatidylinositol (GPI) -immobilized single chain glycoprotein expressed at high levels on monocytes. Furthermore, the CD14 antibody reacted with some macrophages. The CD16 antibody reacted with a 50-65 kDa transmembrane IgG Fc receptor (FcgRIII). CD16 antigen was expressed on monocytes, macrophages, granulocytes and NK cells. Monocytes can be divided into two subsets depending on their CD16 expression; resident monocytes are CD ⁺ CD16 ⁻ and inflammatory monocytes are CD14 ^low CD16 ⁺ . 20 ng / ml of MGD-CSF is differentiated to either CD14 ⁺ CD16 ⁻ or CF14 ^low CD16 ⁺ monocytes with 30% differentiation, 100 ng / ml MGD-CSF with 30% differentiation and 500 ng / ml with differentiation Was induced 53%. Positive controls G-CSF, GM-CSF and M-CSF promoted CD14 differentiation by 41%, 38% and 51%, respectively.

(C. Dendritic cell differentiation)
As shown in FIG. 17, MGD-CSF stimulated dendritic cell differentiation from undifferentiated cells to differentiated dendritic cells carrying CD86 and CD1 markers. As a negative control, the baseline level of dendritic cell differentiation in the presence of empty vector was determined to be 4%. MGD-CSF stimulated 22% of undifferentiated cells to differentiate into dendritic cells.

(Example 11: MGD-CSF promotes bone marrow colony formation)
To evaluate the stimulatory effect of MGD-CSF on human bone marrow derived myeloid cells (CFU-G, CFU-M and CFU-GM) precursor proliferation, a colony formation assay was performed. Positive controls for stimulation of bone marrow precursors were 0.1 ng / ml of G-CSF, 1 ng / ml and 10 ng / ml and 0.01 ng / ml, 0.1 μg / ml and 3 ng / ml of GM-CSF. It was addition. MGD-CSF protein was diluted to a final concentration of 20, 100 and 500 ng / ml in each test concentration of methylcellulose-based medium. Cells were added so that each three replicate cultures contained 3 × 10 ⁴ cells. Replicate cultures are incubated for 14 days at 37 ° C., 5% CO ₂ , then counted, photographed, classified based on morphology as CFU-G, CFU-M or CFU-GM, and FACS Analysis was carried out. Statistical analysis was performed to assess changes in colony number, size and morphology. MGD-CSF promoted the formation of CFU-G, CFU-M, CFU-GM, and total colony formation. Furthermore, as described further below, MGD-CSF promoted large CFU-M colonies different from those promoted by G-CSF or GM-CSF. These results suggest that MGD-CSF enhanced early hematopoietic precursor formation.

  FIG. 18A and FIG. 18B show the effect of MGD-CSF on human bone marrow colony formation in the absence of exogenous cytokines. As shown in FIG. 18A, purified MGD-CSF had little effect on granulocyte colony formation (CFU-G) compared to G-CSF and GM-CSF. MGD-CSF stimulated monocyte colony formation (CFU-M) in a dose-dependent manner. As shown in FIG. 18B, MGD-CSF stimulated granulocyte-monocyte colony forming CFU-GM in a dose-dependent manner. MGD-CSF also stimulated colony forming capacity (CFC) in a dose-dependent manner. FIG. 18C shows the effect of MGD-CSF on human bone marrow colony formation in the presence of cytokine IL-3 and stem cell factor (SCF). Under these conditions, purified MGD-CSF stimulated CFU-G, CFU-GM, CFU-M and total CFC in a dose-dependent manner. The distribution of bone marrow precursors in the presence of MGD-CSF was different from that of G-CSF or GM-CSF.

(Example 12: Profile of biological activity of MGD-CSF)
As shown in FIG. 19, MGD-CSF is treated with non-activated B cell proliferation (BPro4), the ability to stimulate glucose uptake by adipocytes (Gu2Gy3T3), inactivated monocyte proliferation (MonPro4), NK cell proliferation and / or Or in assays that measure survival (NKGlo), T cell proliferation (TPro4), activated primary B cell proliferation (aBPro4), activated primary monocyte proliferation (aMonPro3), and activated primary T cell proliferation (aTPro4). The biological effect was tested. Results were considered significant if at least 2 standard deviations from the median. MGD-CSF does not stimulate the proliferation of activated monocytes, nor does it stimulate the proliferation of activated or inactivated B cells or T cells. Proliferation was specifically stimulated.

(Example 13: Profile of MGD-CSF-induced cytokine secretion)
As shown in FIG. 20, MGD-CSF was tested for its biological effect on cytokine secretion from NK cells. The assay was performed as described in Example 11. According to the manufacturer's instructions, Linco, Inc. Conditioned media was removed at the end of the experiment to determine the type and amount of cytokine secretion using the Luminex cytokine assay kit from (St. Charles, MO). Results were considered significant if at least 2 standard deviations from the median. MGD-CSF stimulated secretion of GM-CSF, IL-12 and IL-13.

(Example 14: MGD-CSF stimulated CFU-M differentiation from HSC cells)
MGD-CSF increased the size and number of bone marrow colonies formed from human bone marrow cells in a dose-dependent manner. The upper left panel of FIG. 21 shows representative photographs of CFU-M colonies observed in bone marrow cells cultured in the absence of cytokines (buffer). There is weak or no evidence of colonization. The upper middle panel shows a representative picture of CFU-M colonies induced by GM-CSF; colony formation was evident. The upper right panel shows representative photographs showing that G-CSF does not stimulate CFU-M formation. The bottom three panels of FIG. 16 show representative photographs of CFU-M induced by MGD-CSF. Both number and size increased in a dose-dependent manner from 20 ng / ml to 500 ng / ml MGD-CSF. The cells were examined and photographed with an Axiovert 25 microscope and AxioCam HRc (both Carl Zeiss, Gottingen, Germany) using a 40 × lens. The cell is Zeiss
Visualized with a KS300 3.0 digital imaging system.

  The size of colonies induced by MGD-CSF was larger than the size of colonies induced by GM-CSF. About 10% of the colonies induced by MGD-CSF were very large, ranging from 100 to 2000 microns. MGD-CSF induced these large colonies both in the presence and absence of the cytokines SCF and IL-3. These data indicate that MGD-CSF promoted the formation of early bone marrow precursors, including earlier precursors than GM-CSF or G-CSF. They also suggest that MGD-CSF promotes differentiation of either the M1 or M2 macrophage lineage or both.

(Example 15: MGD-CSF stimulated HSC differentiation into dendritic cells)
Human bone marrow CD34 + cells (Cambrex, Inc., Baltimore MD) were plated in serum-free X-vivo 20 medium (Cambrex, Inc., Baltimore MD) on 24-well cell culture plates and conditioned medium for vector control (Cambrex, Inc., Baltimore MD). CM) or MGD-CSF CM. The cells were examined and photographed with an Axiovert 25 microscope and AxioCam HRc (both Carl Zeiss, Gottingen, Germany) using a 40 × lens. The cell is Zeiss
Visualized with a KS300 3.0 digital imaging system.

As shown in FIG. 22, an increase in the number of elongated cells was observed in MGD-CSF treated cultures with increasing MGD-CSF concentration. Differentiation into dendritic cells was confirmed using an anti-CD1a antibody. Treatment of the culture for 2 weeks with MGD-CSF induced differentiation of human bone marrow CD34 ⁺ into 20% CD1-positive dendritic cells. MGD-CSF induced dendritic cell differentiation from human bone marrow cells in a dose-dependent manner (FIG. 22). The cells shown in all four panels were examined and photographed with an Axiovert 25 microscope and AxioCam HRc (both Carl Zeiss, Gottingen, Germany) using a 40 × lens. Cells were visualized with a Zeiss KS300 3.0 digital imaging system.

  The upper left panel of FIG. 22 shows representative photographs of bone marrow cells cultured in the absence of cytokines (medium). Cells are typically small and rounded. The upper right panel shows a representative picture of bone marrow cells that are elongated and flattened to more closely resemble dendritic cells in response to 20 mg / ml MGD-CSF. The lower left panel shows representative photographs demonstrating that MGD-CSF as high as 100 ng / ml has a more pronounced effect on HSC cell morphology. The cells are larger and elongated. The lower right panel shows that MGD-CSF at a concentration of 500 ng / ml produces large, flat, elongated cells with a morphological appearance of dendritic cells.

(Example 16: MGD-CSF gene expression)
Various levels of gene expression were obtained using the Affymetrix GeneChip® array platform with probe 237046_x_at, Human Genoma U133 and U133Plus_2 (Affymetrix, Inc, Santa Clara, Calif.) Using the GeneLogiology database of the original Logiology database. By individual human cancer tissue specimens. This also includes probes PRB107386 and PRB107386_at. To use Five Prime
Comparison was made by interrogating microarray chips designed by Therapeutics. These probes were used to confirm the expression of MGD-CSF in the tissues of patients with hyperproliferative hematological abnormalities. This analysis identified different gene expression patterns between different tissue types and different tissue stages. Table 8, third column lists the number of disease specimens tested positive (MGC34647 positive) for the presence of MGC34647. The fourth column of Table 8 lists the number of specimens examined (Total Gene Logic). MGD-CSF was expressed in most patients with myelodysplastic syndrome. The percentage of patients expressing MGD-CSF varied with the observed pathology and was highest in patients with refractory anemia with excess blasts or cyclic iron blasts. Half of patients with acute B cell lymphoblastic leukemia expressed MGD-CSF. A subset of patients with acute myeloid leukemia expressed MGD-CSF. This percentage varied between 14-25% depending on the pathological symptoms of the disease. MGD-CSF was generally not expressed in patients with chronic myelogenous leukemia, chronic lymphocytic leukemia, or acute promyelocytic leukemia.

(Sequence Listing)
This application attaches a sequence listing in the form of a sequence listing delivery sheet and paper.

Claims (5)

A composition for modulating an immune response in a subject comprising:
(A) a polypeptide comprising any one of the amino acid sequences of SEQ ID NO: 7, 9 or 10 and having an activity of stimulating the proliferation of immune cells, and (b) an amino acid sequence of any of SEQ ID NO: 7, 9 or 10 An antibody specific for a polypeptide selected from polypeptides having an amino acid sequence that is at least 95% identical to and having the activity of stimulating the proliferation of immune cells, wherein the modulation of the immune response is to suppress inflammation and A composition that is / or suppression of an autoimmune disease. The composition of claim 1, wherein the antibody is selected from a monoclonal antibody, a polyclonal antibody , a single chain antibody and an active fragment of an antibody. The modulation of immune responses, rheumatoid arthritis, osteoarthritis, psoriasis, inflammatory bowel disease, multiple sclerosis, treatment or prevention of a disease selected from contact and fulminant hepatic failure, according to claim 1 Composition. The composition according to claim 1 or 2, for administration to a patient with an inflammatory disease. The composition according to claim 1 or 2, for administration to a patient with an autoimmune disease.